Amino acid containing thapsigargin analogues deplete androgen receptor protein via synthesis inhibition and induce the death of prostate cancer cells

Abstract

There are quantitative and/or qualitative mechanisms allowing androgen receptor (AR) growth signaling in androgen ablation refractory prostate cancer cells. Regardless of the mechanism, agents that deplete AR protein expression prevent such AR growth signaling. Thapsigargin (TG) is a highly cell-penetrant sequiterpene-lactone that once inside cells inhibits (IC(50), ∼ 10 nmol/L) critically important housekeeping SERCA 2b calcium pumps in the endoplasmic reticulum. Using a series of five genetically diverse androgen ablation refractory human prostate cancer lines (LNCaP, LAPC-4, VCaP, MDA-PCa-2b, and CWR22Rv1), TG inhibition of SERCA pumps consistently results in depletion of the endoplasmic reticulum Ca(+2) coupled with μmol/L elevation in the intracellular free Ca(+2) initiating a molecular cascade that: (a) inhibits Cap-dependent AR protein synthesis resulting in 90% depletion of AR protein by 24 hours of TG exposure, (b) arrests the cells in G(0), and (c) induces their apoptotic death. Unfortunately, due to its highly lipophilic nature, TG is not deliverable as a systemic agent without host toxicity. Therefore, TG analogues containing amino acids were developed, which retain ability to deplete AR protein and induce cell death and which can be covalently linked to peptide carriers producing water soluble prodrugs for systemic delivery. Specific amino acid sequences are used to restrict the liberation of cytotoxic amino acid containing TG analogues from the peptide prodrug by prostate-specific proteases, such as prostate-specific antigen and prostate-specific membrane antigen, or cancer-specific proteases, such as fibroblast activation protein, so that toxicity of these prodrugs is selectively targeted to metastatic sites of prostate cancer. Based on these results, these prodrugs are undergoing clinical development.

Figure 1

A, chemical Structure for TG, its amine-containing 8-O-(12-dodecanyl)-8-O-debutanoyl TG analogue (12ADT), and its amino-acid analogues (i.e., Alanyl-12ADT, Aspartyl-12ADT, and Leucyl-12ADT). B, amino acid sequences used to restrict TG analogues from the peptide prodrugto prostate-specific proteases, prostate-specific antigen, fibroblast activation protein, and prostate-specific membrane antigen. Mu, morpholino protection group; AA, amino acid.

Figure 2

A, changes in the level of indicated proteins in LNCaP cells during the first 24 h of exposure to 500 nmol/L TG. B, changes in the level of indicated proteins in CWR22Rv1 cells duringthe first 48 h of exposure to 500 nmol/L Leu-12ADT. C, translocation of apoptosis-inducingfactor protein from mitochondria to nucleus of LNCaP and LAPC-4 prostate cancer cells by 48-h exposure to 500 nmol/L TG. Left, untreated cells. Right, 12ADT-treated cell. D, XBP-1 processingin LNCaP prostate cancer cells treated with 500 nmol/L leucyl-12ADT (Leu-12ADT) for 24 h. LNCaP cells were transfected with an expression construct encoding a FLAG-tagged form of the XBP-1 gene fused to venus-GFP so that a fluorescent protein can only be produced when the fusion gene is processed by IRE1. Under non-ER–stressed conditions, the transgenic transcript is not spliced, and therefore, its translation is terminated at the stop codon between FLAG-tagged XBP-1-ΔDBD and the venus gene so that no fluorescent protein is produced. In contrast, during ER stress, the activated IRE-1 slices out a 26-nt intron in the XBP-1 portion, resultingin a frame shift of the fusion mRNA, allowingthe synthesis of a 45-kDa FLAG-tagged XBP-1-ΔDBD/venus fusion fluorescent protein. Thus, flow cytometric analysis of GFP expression documents that IRE1-dependent XBP-1 processing occurs in all cells within 24 h.

Figure 3

Relative level of AR protein in the various androgen ablation refractory human prostate cancer cell lines compared with each other versus normal human prostate stromal cells (PrSCs). Values below the upper gel are the relative expression normalized to LNCaP cells. In the second blot, the level of AR in 5,000 LNCaP cells is compared with 5,000 to 200,000 prostate stromal cells. Kinetics of the depletion of AR protein in the various human prostate cancer lines after exposure to 500 nmol/L TG. Values below the each gel are the relative expression normalized to control (unexposed) cells for each line.

Figure 4

AR mRNA expression by indicated cell lines at various times after exposure to 1 µmol/L TG. Kinetics of the decrease in the rate of AR-specific protein synthesis versus total protein synthesis induced in LNCaP cells exposure to 1 µmol/L TG. Change in the level of cleaved PARP, phosphorylated elF4E, phosphorylated 4E-BP1, and elF4G protein in LNCaP cells exposed for 24 h to 1 µmol/L TG.