L. Shatkina

The Cochaperone Bag-1L Enhances Androgen Receptor Action via Interaction with the NH2-Terminal Region of the Receptor

ABSTRACT Members of the Bag-1 family of cochaperones regulate diverse cellular processes including the action of steroid hormone receptors. The largest member of this family, Bag-1L, enhances the transactivation function of the androgen receptor. This occurs primarily through interaction with the NH2 and COOH termini of the receptor. At the NH2 terminus of the receptor, Bag-1L interacts with a region termed τ5. Bag-1M, a naturally occurring variant of Bag-1L that binds to τ5 but is defective in the COOH-terminal interaction, is less efficient in enhancing the transactivation function of the receptor. Surface plasmon resonance and transfection studies showed that the molecular chaperone Hsp70 contributes to the binding of Bag-1L to τ5 and to the regulation of the transactivation function of the androgen receptor. Chromatin immunoprecipitation studies demonstrated that the androgen receptor, Hsp70, and Bag-1L are all targeted to the androgen response elements of the gene that encodes prostate-specific antigen. These studies demonstrate the regulation of transcriptional activity of androgen receptor by a molecular chaperone-cochaperone complex.

A peptidic unconjugated GRP78/BiP ligand modulates the unfolded protein response and induces prostate cancer cell death.

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.

Specific N-terminal mutations in the human androgen receptor induce cytotoxicity

Polyglutamine (polyQ) stretch amplification in different proteins causes neurodegenerative disease. These proteins form intracellular aggregates thought to be cytotoxic but differ in pathology and tissue specificity. Here, we demonstrate that specific sequences outside the polyQ stretch of the human androgen receptor contribute to polyQ pathology. An exchange of two N-terminal serine phosphorylation residues to alanine in the wild type androgen receptor (ARQ22dm) resulted in cytoplasmic accumulation and increased early hormone-dependent aggregation of the receptor. In a Drosophila model, the ARQ22dm was cytotoxic, and developing larvae expressing this receptor showed behavioral abnormalities and severely impaired locomotion. In contrast, the same double mutation in an androgen receptor with an extended polyQ stretch was less toxic. The response of the receptors to inhibitors of polyglutamine toxicity is altered by the amino acid exchanges suggesting that careful consideration is needed in the choice of potential therapies of disorders involving toxic polyQ species.

Development of Bag-1L as a therapeutic target in androgen receptor-dependent prostate cancer

Targeting the activation function-1 (AF-1) domain located in the N-terminus of the androgen receptor (AR) is an attractive therapeutic alternative to the current approaches to inhibit AR action in prostate cancer (PCa). Here we show that the AR AF-1 is bound by the cochaperone Bag-1L. Mutations in the AR interaction domain or loss of Bag-1L abrogate AR signaling and reduce PCa growth. Clinically, Bag-1L protein levels increase with progression to castration-resistant PCa (CRPC) and high levels of Bag-1L in primary PCa associate with a reduced clinical benefit from abiraterone when these tumors progress. Intriguingly, residues in Bag-1L important for its interaction with the AR AF-1 are within a potentially druggable pocket, implicating Bag-1L as a potential therapeutic target in PCa.

Membrane Localization and Rapid Non-Transcriptional Action of the Androgen Receptor

Androgens play a major role in male sexual development and in prostate growth during later life (for a review, see Brinkman 2001). They function by binding a cytoplasmic androgen receptor (AR) that is present in target cells in an inactive state in association with molecular chaperones and co-chaperones. Upon hormone binding, the receptor is thought to dissociate from the inactivating proteins and is transported into the nucleus where it binds discrete nucleotide sequences to modulate the expression of specific genes. In the nucleus, the AR also negatively regulates the expression of genes via protein-protein interaction (for review see Cato and Peterziel, 1998).

Abstract A216: Distinct peptides derived from the cochaperone Bag1‐L inhibit prostate cancer cell growth

Introduction and objectives: Bag1 (Bcl‐2 associated athanogene‐1) is a family of proteins conserved throughout evolution, promoting cell survival and inhibiting apoptosis under stressful conditions. They are found overexpressed in several types of tumors and associated with poor prognosis. Intriguingly, the nuclear isoform of the Bag‐1 proteins, Bag‐1L, also exhibits pro‐apoptotic properties through an unknown mechanism. In extensive deletion mutagenesis studies we identified sequences in the Bag‐1L protein that exert pro‐apoptotic action. The objective of the study is to characterize the mechanism of action of these sequences and to explore their use as candidate peptides for prostate cancer therapy. Materials and Methods: In vitro inhibition of tumor cell growth was analyzed in clonogenic assay where prostate cells were transfected with plasmids expressing different Bag‐1L peptides. In vivo growth inhibition was analysed by the injection of prostate cells stably expressing the Bag‐1L peptides into the flank of athymic nude mice and analyzing their effect on tumor size in vivo. Histological analysis for apoptosis was carried out on the tumor samples. For analyzing the interaction partners of the Bag‐1L peptides, the peptides were generated as fusion proteins with glutathione S transferase (GST), immobilized on glutathione agarose beads and allowed to interact with lysates from prostate cells. Specifically bound proteins were then eluted and analysed by mass spectrometry and by Western blotting. Results: Overexpression of a 68 amino acid sequence of Bag‐1L reduced the growth of 22Rv.1 prostate cancer cells but not of benign prostate hyperplasia cells in a clonogenic assay. Stable overexpression of this peptide in the 22Rv.1 prostate cancer cells also reduced cell growth in vivo in athymic nude mice. Histological analysis revealed massive apoptosis in the tumor cells expressing the peptide. Further deletion analysis of the 68 amino acid peptide showed that the N‐terminal 40 amino acids but not the C-terminal 29 amino acids are sufficient for the growth inhibitory action. Protein‐protein interaction studies using GST‐pull down assay demonstrated that the growth inhibitory function of the peptides correlated with their ability to bind glucose regulated proteins 75 and 78 (GRP75 and GRP78). GRP75 and GRP78 have been implicated in tumor formation through their action in the mitochondria and in the endoplasmic reticulum in the regulation of the unfolded protein response. Conclusions: We have therefore identified Bag1‐L peptides which when expressed in prostate tumor cells reduce the growth in vitro as well as in a mouse model of tumor formation. The action of the peptides is to induce apoptosis and it is correlated with their ability to bind the molecular chaperones GRP75 and GRP78. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A216.