Targeting carcinoma-associated fibroblasts within the tumor stroma with a fibroblast activation protein-activated prodrug

Background Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG. Methods The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9-11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided. Results FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC(50) = 3.5nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206mm(3), 95% CI = 0.068 to 0.344mm(3); control: mean = 0.580mm(3), 95% CI = 0.267 to 0.893mm(3)) and 0.24 (treated: mean = 0.131mm(3), 95% CI = 0.09 to 0.180mm(3); control: mean = 0.543mm(3), 95% CI = 0.173 to 0.913mm(3)), respectively, on day 21 after therapy. Conclusions This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.     

Engineering enzymatically activated "molecular grenades" for cancer

Animal toxicology and clinical phase I trial data have documented that the PSMA-activated thapsigargin drug, G202 is non-myelosuppressive. This lack of myelosuppression facilitates G202 combination with a variety of additional clinically approved drugs. For example, thapsigargin's ability to induce ER stress raises its potential for synergy when combined with radiation and cytotoxic chemotherapies. Finally, G202-based delivery of the thapsigargin analog causes marked reduction of expression of the androgen receptor (AR) in prostate cancer cells and the estrogen receptor (ER) protein in breast cancer. These results suggest that combination therapy with anti-androgen/estrogens and G202 could be synergistic against prostate and/or breast cancer. These combinatorial approaches are currently under pre-clinical evaluation in our laboratories.     

Bipolar androgen therapy: The rationale for rapid cycling of supraphysiologic androgen/ablation in men with castration resistant prostate cancer

Androgen ablation is highly effective palliative therapy for metastatic prostate cancer but eventually all men relapse. New findings demonstrating that androgen receptor (AR) expression continues in androgen ablated patients has resulted in the classification “Castration Resistant Prostate Cancer” (CRPC) and has led to the development of new second‐line “anti‐ligand” hormonal agents. In this background is the paradoxical observation that the growth of some AR‐expressing “androgen sensitive” human prostate cancer cells can be inhibited by supraphysiologic levels of androgens. This response may be due to effects of high‐dose androgen on inhibiting re‐licensing of DNA in cells expressing high levels of AR. It may also be due to recently described effects of androgen in inducing double strand DNA breaks. Based on available preclinical data described in this review demonstrating the effects of supraphysiologic levels of testosterone on inhibition of growth of CRPC xenografts, we initiated a clinical trial in men with CRPC testing the effect of monthly treatments with an intramuscular (IM) depot injection of testosterone. This IM formulation achieves supraphysiologic levels of testosterone that cannot be achieved with standard testosterone gel‐based applications. The supraphysiologic testosterone level is followed by a rapid drop to castrate levels of testosterone with each cycle of therapy. This “bipolar androgen therapy” will not allow time for prostate cancer cells to adapt their AR expression in response to environmental conditions. The goal is to determine if a clinical response can be achieved through this non‐adaptive rapid cycling approach in men with CRPC. Prostate 70: 1600–1607, 2010. © 2010 Wiley‐Liss, Inc.     

A dimeric peptide that binds selectively to prostate-specific membrane antigen and inhibits its enzymatic activity

Prostate-specific membrane antigen (PSMA) is highly expressed by both normal and malignant prostate epithelial cells and by the neovasculature of many tumor types; however, it is not expressed by normal endothelial cells or other normal tissues. PSMA, therefore, represents an attractive candidate for selectively targeted therapies for prostate and/or other solid tumors. As an alternative approach to antibody-based anti-PSMA therapies, small peptides that bind selectively to PSMA-producing cells can be used to deliver cytotoxic drugs, protein toxins, and viruses selectively to malignant sites while minimizing systemic toxicity to normal tissues. Small peptides are relatively inexpensive to produce, not immunogenic, and easily coupled to cytotoxic agents. In the present study, a random phage library consisting of linear 12 amino acid peptides was used to identify peptides that bound selectively to PSMA. From a series of monomeric peptides, one with the sequence WQPDTAHHWATL was used to show binding of soluble peptide to PSMA. A dimeric version of this peptide showed markedly enhanced binding to soluble PSMA and an IC50 of 2.2 micromol/L for inhibition of PSMA enzymatic activity. Fluorescently labeled dimeric peptide bound selectively to PSMA-producing prostate cancer cells in vitro with no significant binding to non-PSMA-producing cells. Molecular modeling of the dimeric peptide revealed that histidine residues in close vicinity can efficiently coordinate a divalent ion and hold the peptide in a favorable configuration for binding and subsequent inhibition. These dimeric peptides, therefore, represent putative PSMA-selective targeting agents that are currently being evaluated for selective binding in vivo.     

Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer

Cells possess within their epigenetic repertoire the ability to undergo an active process of cellular suicide termed programmed (or apoptotic) cell death. This programmed cell death process involves an epigenetic reprogramming of the cell that results in an energy-dependent cascade of biochemical and morphologic changes (also termed apoptosis) within the cell, resulting in its death and elimination. Although the final steps (i.e., DNA and cellular fragmentation) are common to cells undergoing programmed cell death, the activation of this death process is initiated either by sufficient injury to the cell induced by various exogenous damaging agents (e.g., radiation, chemicals, viruses) or by changes in the levels of a series of endogenous signals (e.g., hormones and growth/survival factors). Within the prostate, androgens are capable of both stimulating proliferation as well as inhibiting the rate of the glandular epithelial cell death. Androgen withdrawal triggers the programmed cell death pathway in both normal prostate glandular epithelia and androgen-dependent prostate cancer cells. Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen ablation; however, they do retain the cellular machinery necessary to activate the programmed cell death cascade when sufficiently damaged by exogenous agents. In the normal prostate epithelium, cell proliferation is balanced by an equal rate of programmed cell death, such that neither involution nor overgrowth normal occurs. In prostatic cancer, however, this balance is lost, such that there is greater proliferation than death producing continuous net growth. Thus, an imbalance in programmed cell death must occur during prostatic cancer progression. The goal of effective therapy for prostatic cancer, therefore, is to correct this imbalance. Unfortunately, this has not been achieved and metastatic prostatic cancer is still a lethal disease for which no curative therapy is currently available. In order to develop such effective therapy, an understanding of the programmed death pathway, and what controls it, is critical. Thus, a review of the present state of knowledge concerning programmed cell death of normal and malignant prostatic cells will be presented. © 1996 Wiley-Liss, Inc.     

A combinatorial approach to the selective capture of circulating malignant epithelial cells by peptide ligands

Early detection is critical in the administration of definitive and curative therapy of cancer. However, current detection methods are ineffective at identifying the presence of circulating metastatic cancer cells in the blood because they typically sample only a relatively small volume of blood. One strategy for sampling larger blood volumes would be to capture circulating cells in vivo over an extended period of time. The development of such a method would be substantially facilitated by the identification of peptide ligands that bind selectively to metastatic cancer cells in the blood with high affinity. To identify such ligands a combinatorial peptide library was synthesized on polyethylene acrylamide (PEGA) resin and screened for binding to malignant epithelial cells. Using Biacore, cell binding assays were performed to demonstrate that peptides selected from PEGA bead screen can bind selectively to malignant epithelial cancer cells and not to circulating leukocytes under physiologic shear stress conditions. One peptide, with the sequence QMARIPKRLARH, was used to demonstrate selective labeling of malignant epithelial cells spiked in whole blood. When immobilized on appropriate surfaces, these peptides could be used in both in vivo and ex vivo cell separation devices to efficiently and selectively capture metastatic epithelial cancer cells from flowing blood.     

Design, synthesis, and pharmacological evaluation of thapsigargin analogues for targeting apoptosis to prostatic cancer cells.

A series of thapsigargin (TG) analogues, containing an amino acid applicable for conjugation to a peptide specifically cleaved by prostate-specific antigen (PSA), has been prepared to develop the drug-moiety of prodrugs for treatment of prostatic cancer. The analogues were synthesized by converting TG into O-8-debutanoylthapsigargin (DBTG) and esterifying O-8 of DBTG with various amino acid linkers. The compounds were evaluated for their ability to elevate the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in TSU-Pr1 cells, their ability to inhibit the rabbit skeletal muscle SERCA pump, and their ability to induce apoptosis in TSU-Pr1 human prostatic cancer cells. The activity of analogues, in which DBTG were esterified with omega-amino acids [HOOC(CH(2))(n)()NH(2), n = 5-7, 10, 11], increased with the linker length. Analogues with 3-[4-(L-leucinoylamino)phenyl]propanoyl, 6-(L-leucinoylamino)hexanoyl, and 12-(L-serinoylamino)dodecanoyl were considerably less active than TG, and analogues with 12-(L-alaninoylamino)dodecanoyl and 12-(L-phenylalaninoylamino)dodecanoyl were almost as active as TG. The 12-(L-leucinoylamino)dodecanoyl gave an analogue equipotent with TG, making this compound promising as the drug-moiety of a PSA sensitive prodrug of TG.     

A prostate-specific antigen activated N-(2-hydroxypropyl) methacrylamide copolymer prodrug as dual-targeted therapy for prostate cancer

Prostate cancer targeted peptide prodrugs that are activated by the serine protease activity of prostate-specific antigen (PSA) are under development in our laboratory. To enhance delivery and solubility of these prodrugs, macromolecular carriers consisting of N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers were covalently coupled to a PSA-activated peptide prodrug. HPMA copolymers are water-soluble, nonimmunogenic synthetic carriers that exhibit promise for drug delivery applications. These macromolecular copolymers enter the interstitium of solid tumors by the enhanced permeability and retention effect. The PSA-activated peptide substrate imparts selectivity because it is specifically hydrolyzed to release a cytotoxin at the site of prostate tumor. Enzymatically active PSA is present in high amounts in the extracellular fluid of a tumor, but PSA is inactivated in blood by binding to serum protease inhibitors. As an initial proof of concept, the HPMA copolymer was synthesized with a peptide substrate (HSSKLQ) bound to a fluorophore, 7-amino-4-methylcoumarin (AMC). PSA cleavage of the HPMA-HSSKLQ-AMC copolymer was observed, which led to the synthesis of an HPMA-based copolymer with the prodrug SSKYQ-L12ADT [HPMA-morpholinocarbonyl-Ser-Ser-Lys-Tyr-Gln-Leu-12-aminododecanoyl thapsigargin (JHPD)]. L12ADT is a potent analogue of the highly cytotoxic natural product thapsigargin. HPMA-JHPD was hydrolyzed by PSA in vitro and was toxic to prostate cancer cells in the presence of active PSA. The HPMA-JHPD produced no systemic toxicity when given at a 500 micromol/L L12ADT equivalent dose. Analysis of tumor tissue from mice treated with a single or multiple dose of the HPMA-JHPD copolymer showed release and accumulation of the L12ADT toxin within the tumor tissue.