Cor A. Berrevoets

The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens

The human prostate tumor cell line LNCaP contains an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induce reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.

Mechanisms of androgen receptor activation and function.

Androgens play a crucial role in several stages of male development and in the maintenance of the male phenotype. Androgens act in their target cells via an interaction with the androgen receptor, resulting in direct regulation of gene expression. The androgen receptor is a phosphoprotein and modulation of the phosphorylation status of the receptor influences ligand-binding and consequently transcription activation of androgen responsive genes. Androgen binding induces a conformational change in the ligand-binding domain, accompanied by additional receptor phosphorylation. Subsequently the liganded androgen receptor interacts with specific androgen response elements in the regulatory regions of androgen target genes, resulting in stimulation of gene expression. Anti-androgens induce a different conformational change of the ligand-binding domain, which does not or only partially result in stimulation of transactivation. Interestingly, different anti-androgens can induce different inactive conformations of the androgen receptor ligand-binding domain. Recent evidence strongly supports a ligand dependent functional interaction between the ligand-binding domain and the NH2-terminal transactivating domain of the androgen receptor. Two regions in the NH2-terminal domain are involved in this interaction, whereas in the ligand-binding domain the AF-2 AD core region is involved.

Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy.

The human androgen receptor (hAR) is an important regulatory protein particularly in male sexual differentiation. The investigation of hAR functionality has been hampered by the lack of AR specific monoclonal antibodies recognizing the functional domains of the receptor. Therefore production of high affinity mono-specific polyclonal (PAbs) and monoclonal antibodies (MAbs) directed to the hAR was initiated following the synthetic peptide (SP) strategy. Five hAR specific peptides were selected on the basis of their predicted antigenic properties avoiding homology with other steroid hormone receptors. Peptide specific polyclonal antisera were obtained following selected immunization protocols. Mono-specific polyclonal antibody responses were elicited to all peptides in mice and rabbits. Crossreactivity of the peptide specific antisera with the native hAR in various biochemical assays was observed with two out of five peptides. Peptide SP61 (hAR residues 301-320) was used for the generation site-directed MAbs specific for the hAR. Specificity for the hAR was established by immunoprecipitation, immune-complex density gradient centrifugation and immunohistochemistry on human prostate tissue sections. The multi-assay performance of the selected high affinity antibodies proved the usefulness of the straight forward peptide approach and opens a wide field of possible biochemical and physiological investigations into questions related to androgen action.

EFFECTS OF ANTIANDROGENS ON TRANSFORMATION AND TRANSCRIPTION ACTIVATION OF WILD-TYPE AND MUTATED (LNCaP) ANDROGEN RECEPTORS

LNCaP cells contain androgen receptors with a mutation in the steroid binding domain (Thr 868 changed to Ala) resulting in a changed hormone specificity. Both the wild-type and mutated androgen receptors were transfected into COS cells. Transcription activation was studied in cells co-transfected with an androgen sensitive reporter (CAT) gene. The wild-type androgen receptor was activated by the agonist R1881, but the antiandrogens did not enhance transcription apart from a partial agonistic effect at high concentrations of cyproterone acetate. The mutated androgen receptor was fully activated by R1881, cyproterone acetate and hydroxyflutamide, but not by ICI 176,334. Receptor transformation to a tight nuclear binding state was studied by preparation of detergent washed nuclei and Western blotting with a specific antibody against the androgen receptor. Nuclei of COS cells transfected with wild-type receptor retained the receptor when the cells had been treated with the agonist R1881, partially retained receptors when treated with antiandrogen cyproterone acetate, but did not retain receptor when treated with hydroxyflutamide or ICI 176,334. The cells transfected with the mutated receptor additionally retained nuclear receptors after treatment with hydroxyflutamide. We conclude that each one of the three antiandrogens tested displayed different characteristics with respect to its effect on transformation and transcription activation.

Antiandrogens: selective androgen receptor modulators

Antiandrogens can efficiently block androgen receptor (AR) mediated gene expression, and are therefore useful tools in the treatment of androgen dependent prostate cancer. Antiandrogens are either complete or partial inhibitors of AR activity, depending on the nature of the compound. As compared to androgens, antiandrogens induce a different AR conformation, thereby influencing the recruitment of co-regulators (coactivators and corepressors). This ligand-selective modulation of AR activity is affected by an AR mutation (Thr877Ala substitution) found in prostate cancer. In contrast to the wild-type AR, the mutant AR conformation induced by cyproterone acetate (CPA) and hydroxyflutamide (OHF) is comparable to that induced by androgens. As a consequence, this might affect recruitment of co-regulators, thereby allowing CPA and OHF to act as strong agonists on the mutant AR.

Studies on the human prostatic cancer cell line LNCaP

The effects of androgens, antiandrogens, and other steroid hormones on growth of the human prostate cancer cell line LNCaP were studied. Despite the absence of receptors for progesterone and estradiol, the growth rate of the androgen responsive LNCaP-FGC cells increased when cultured in the presence of either estrogens or progestagens. In addition, most antiandrogens were also growth stimulators. This aberrant response was due to a threonine to alanine substitution at amino acid position 868 in the steroid binding domain of the androgen receptor (AR). Only the antiandrogen ICI 176,334 could block transcription and cell growth by the mutant receptor. By immunoprecipitation of the AR from LNCaP cells with the specific antibody F39.4.1 and Western blotting, three types of heat-shock proteins co-precipitated: hsp90, hsp70 and hsp56. This co-isolation could be prevented by pre-incubating the cells with androgens or with the antiandrogen hydroxyflutamide. Only the antiandrogen ICI 176,334 could block the effect of androgens on complex dissociation and prevent tight nuclear binding of the AR. Hydroxyflutamide could only inhibit tight nuclear binding of the wild-type AR. Therefore, in LNCaP cells the mutation in the steroid binding domain of the AR prevents a blockade of receptor function by most antiandrogens, but not by ICI 176,334, probably because of a different mechanism by which this compound blocks receptor function.

Differential modulation of androgen receptor transcriptional activity by the nuclear receptor co-repressor (N-CoR).

Antiandrogens are widely used agents in the treatment of prostate cancer, as inhibitors of AR (androgen receptor) action. Although the precise mechanism of antiandrogen action is not yet elucidated, recent studies indicate the involvement of nuclear receptor co-repressors. In the present study, the regulation of AR transcriptional activity by N-CoR (nuclear receptor co-repressor), in the presence of different ligands, has been investigated. Increasing levels of N-CoR differentially affected the transcriptional activity of AR occupied with either agonistic or antagonistic ligands. Small amounts of co-transfected N-CoR repressed CPA (cyproterone acetate)- and mifepristone (RU486)-mediated AR activity, but did not affect agonist (R1881)-induced AR activity. Larger amounts of co-transfected N-CoR repressed AR activity for all ligands, and converted the partial agonists CPA and RU486 into strong AR antagonists. In the presence of the agonist R1881, co-expression of the p160 co-activator TIF2 (transcriptional intermediary factor 2) relieved N-CoR repression up to control levels. However, in the presence of RU486 and CPA, TIF2 did not functionally compete with N-CoR, suggesting that antagonist-bound AR has a preference for N-CoR. The AR mutation T877A (Thr877-->Ala), which is frequently found in prostate cancer and affects the ligand-induced conformational change of the AR, considerably reduced the repressive action of N-CoR. The agonistic activities of CPA- and hydroxyflutamide-occupied T877A-AR were hardly affected by N-CoR, whereas TIF2 strongly enhanced their activities. These results indicate that lack of N-CoR action allows these antiandrogens to act as strong agonists on the mutant AR.

Chimeric antigen receptors for t-cell based therapy

The Chimeric Antigen Receptor (CAR) consists of an antibody-derived targeting domain fused with T-cell signaling domains that, when expressed by a T-cell, endows the T-cell with antigen specificity determined by the targeting domain of the CAR. CARs can potentially redirect the effector functions of a T-cell towards any protein and nonprotein target expressed on the cell surface as long as an antibody or similar targeting domain is available. This strategy thereby avoids the requirement of antigen processing and presentation by the target cell and is applicable to nonclassical T-cell targets like carbohydrates. This circumvention of HLA-restriction means that the CAR T-cell approach can be used as a generic tool broadening the potential of applicability of adoptive T-cell therapy. Proof-of-principle studies focusing upon the investigation of the potency of CAR T-cells have primarily focused upon the genetic modification of human and mouse T-cells for therapy. This chapter focuses upon methods to modify T-cells from both species to generate CAR T-cells for functional testing.

TCRs Genetically Linked to CD28 and CD3ε Do Not Mispair with Endogenous TCR Chains and Mediate Enhanced T Cell Persistence and Anti-Melanoma Activity

Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide–MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.

A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation

A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.