Targeting cath-D in cancer

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Cathepsins have long been known to play an important role in the progression and metastasis of cancer. Cath-D stimulates cancer cell proliferation, fibroblast outgrowth, tumor angiogenesis, and metastasis. In cancer cells, overexpressed cath-D accumulates in cells where it may affect their degradation capacities, and the pro-enzyme is hypersecreted in the tumor micro-environment (Figure 2). Therefore, inhibiting cath-D action requires the development of inhibitors targeting extracellular cath-D, and/or intracellular cath-D located in different parts of the cell (e.g. intracellular vesicles, cytosol, or nucleus).

Inhibitors of cath-D proteolytic activity

In recent years, research interest in the development of potent inhibitors of various aspartic peptidases has arisen, fuelled by the growing evidence of their  involvement in human diseases [131], such as that of renin in hypertension [132], g-secretase in Alzheimer's disease [133], plasmepsins in malaria [134], HIV-1 peptidase in acquired immune deficiency syndrome [135], and secreted aspartic peptidases in Candida infections [136]. As opposed to other proteinases (e.g. serine proteases, metalloproteinases or cysteine cathepsins), no mammalian endogenous lysosomal or cytoplasmic cath-D inhibitor is known to exist. When released into the plasma, cath-D is inactivated by its interaction with a2-macroglobulin at a neutral pH, but not at an acidic pH [137, 138]. Since cath-D requires an acidic pH to be proteolytically active, acidic pH may be the physiological regulator of human cath-D activity. In normal cells, cath-D is only active in acidic intracellular vesicles, and therefore uncontrolled proteolysis is avoided. However, no endogenous cath-D inhibitor is known to exist at acidic pH. It is worth noting that, in cancer, cath-D hypersecreted into the acidic extracellular tumor microenvironment may have a profound effect on matrix remodeling or extracellular factor proteolysis. Most exogenous cath-D inhibitors are synthetic compounds: peptides and polypeptides produced by micro-organisms, plants and lower animals [139, 140]. Organic compounds that esterify the carboxyl group of the Asn33 or Asp231 are synthetic cath-D inhibitors. Studies coupling the complementary methods of combinatorial chemistry and structure-based design, yielded low nanomolar inhibitors of cath-D [141-145]. Cath-D activity is inhibited by structural analogs of synthetic substrates in which an L-amino acid has been replaced by a D-amino acid [139]. The cath-D propeptide segment, which is cleaved off during zymogen activation, has been reported to inhibit pro-cath-D by blocking the active site at neutral pH [24, 25, 146, 147]. At high pH, a stable conformational species of cath-D exists in which the active site is blocked [25]. More recently, a pH-dependent conformational change has been shown to be mediated by electrostatic switches [24]. Peptide fragments derived from the propeptide have been shown to display some inhibitory potency against mature cath-D, suggesting that the development of new classes of pro-peptide-derived inhibitors of cath-D may be promising [147]. Pepstatin A, an inhibitor of aspartic proteases produced by a micro-organism, is the most potent polypeptide inhibitor of  cath-D so far identified [148]. This is a hexa-peptide containing the unusual amino acid, statin (Sta, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid), and has the sequence Iva-Val-Val-Sta-Ala-Sta. It was originally isolated from cultures of various species of Actinomyces due to its ability to inhibit pepsin at picomolar concentrations. It was later found to be a potent inhibitor of nearly all acidic proteases and, as such, has become a valuable research tool. Pepstatin is commonly used to study the role of cath-D in in-vitro systems and in cells. Some studies have seemed to show that pepstatin A administered in vivo induces a significant reduction in the number of metastases, whereas other studies have not confirmed this effect [149]. Inhibition of cath-D by tripeptides containing statin analogs has also been reported [150]. Cath-D polypeptide inhibitors have also been identified in many plants [139], such as tomato leaves [151] and potato tubers [152]. Cath-D inhibitors are also produced by lower animals, such as equistatin from Actinia equina [153, 154] that can also inhibit cysteine cathepsin activity. Interestingly, it has been shown that deoxyribonucleic acids (DNA fragments) can inhibit cath-D proteolytic activity [155].

Inhibitors of cath-D binding activity

Cath-D can also act by protein-protein interaction. Studies of the role of secreted pro-cath-D as a mitogen through its protein binding activity in cancer suggest the involvement of a part of the cath-D profragment (position 27-44) in an interaction with an unknown cell surface receptor [97, 101, 107, 111-113]. Interestingly, an anti-procath-D antibody directed against peptide 27-44 can reverse the growth of human breast tumors in athymic nude mice [111, 112, 156]. Secreted pro-cath-D may also act as a mitogen via its interaction with the M6P moieties of the M6P/IGF-2 receptor, displacing IGF2 from the IGF1 receptor, and leading to the activation the mitogenic IGF1 receptor pathway [109, 110]. We have demonstrated that a mutant ^D231Ncath-D that is devoid of proteolytic activity is still mitogenic for cancer cells and fibroblasts both in vitro in three dimensional (3D) matrices, and in athymic nude mice [53, 105]. These findings suggest that pro-cath-D may act as an extracellular binding protein by directly or indirectly triggering an as-yet unidentified cell surface receptor. Our unpublished results also indicate that pro-cath-D hypersecreted by cancer cells triggers fibroblast invasive growth in a 3D matrix by interacting with a newly-identified fibroblastic cell surface receptor (submitted). The GST pull-down experiments revealed that this novel cath-D receptor binds the 52-, 34- and 14-kDa cath-D fragments, but only poorly to the 4-kDa cath-D profragment, indicating that the interaction interface spans both 34- and 14-kDa cath-D sub-units (submitted). Taken together, these observations suggest the importance of targeting extracellular pro-cath-D, and open new perspectives for the therapeutic inhibition of protease function in cancer by means other than the use of classical catalytic activity inhibitors. Because of the pleotrophic action of secreted cath-D as a binding protein, the best strategy may be to inhibit the extracellular action of pro-cath-D through the use of neutralizing antibodies, rather than by targeting an individual cath-D partner.

Studies in apoptosis also strongly suggest that mature cytosolic cath-D may have an additional role involving protein-protein interaction. So far, no apoptosis-related binding partner of cath‑D has yet been identified. The search for cath-D partners using the yeast­­-two hybrid approach may elucidate the pro-apoptotic function of cath-D independently of its catalytic activity. Our unpublished results using this approach show that cath-D does indeed interact with a pro-apoptotic constituent of the apoptotic pathway. However, it would be premature to envisage blocking the interaction of cath-D using a component of the apoptotic machinery within the cell.

Cath-D substrates in cancer

The discovery of new cath-D physiological substrates is likely to generate new critical targets for cancer therapy. To understand the functions of proteases, it is crucial to identify their substrates. Cath-D cleaves preferentially -Phe-Phe-, -Leu-Tyr-, -Tyr-Leu-, and –Phe-Tyr- bonds in peptide chains containing at least five amino acids at an acidic pH [157]. These peptides contain L-amino acids, and also contain hydrophobic amino acid residues at the site cleaved by cath-D. Recently, proteome-derived database-searchable peptide libraries have been developed to identify endoprotease cleavage sites [158]. This approach may be applicable for cath-D. For a long time the main function of cath-D was thought to be to degrade proteins in lysosomes at an acidic pH. In addition to its established role as a major protein-degrading enzyme in lysosomes and phagosomes, it has been shown that cath-D can also activate precursors of biologically-active proteins, such as prolactin and osteopontin in specialized cells [159-163]. Many cath-D substrates have been reported in vitro, but no endogenous substrates of cath-D in cancer have yet been clearly identified. In proteomics, the set of proteins that can be hydrolyzed by a protease is named its substrate degradome or degradomics [164]. A method termed Terminal Amine Isotopic Labeling of Substrates (TAILS) has recently been developed to identify extracellular and membrane protease substrates using iTRAQ labeling and mass spectrometry [165-167]. This powerful proteomic approach, which permitted the discovery of the MMP-2 substrate degradome [168], can also be applied to the identification of cath-D substrates using cells that do or do not express cath-D.