Roles of cath-D in cancer

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Cath-D is an independent marker of a poor prognosis in breast cancer

In the 1990s, several independent clinical studies showed that the cath-D level in primary breast cancer cytosols is an independent prognostic parameter correlated with the incidence of clinical metastasis and shorter survival times [76]. A meta-analysis of studies on node-negative breast cancer [77], as well as a complete study of 2810 patients in Rotterdam [78], indicate that high concentrations of cath-D are an effective marker of aggressiveness. Cath-D is now recognized as an independent marker of poor prognosis in breast cancer associated with metastatic risk [79]. In recent years, independent studies have confirmed the prognostic value of cath-D in breast cancer [80-88]. The main cath-D producing cells appear to be cancer cells and stromal macrophages [89]. Pro-cath-D is also increased in the plasma of patients with metastatic breast cancer [90-92], indicating that some of the pro-cath-D secreted by tumors can be released into the circulation. Interestingly, proteomic studies have recently confirmed the up-regulation of cath-D in many types of cancer [87, 93, 94].

Cath-D affects multiple steps of cancer progression and metastasis

Cath-D is overexpressed and hypersecreted in a multitude of cancer types (breast cancer, ovarian cancer, endometrial cancer, cancer of the head and neck, bladder cancer, malignant glioma, melanoma). In cancer cells, overexpressed cath-D accumulates in cells where it may affect their degradative capacities, and the pro-enzyme is hypersecreted in the tumor micro-environment (Figure 2). Cath-D hypersecreted by cancer cells may affect stromal cell behavior and/or degrade components of the extracellular matrix, thus modifying the tumor micro-environment (Figure 2).

Several reports have indicated that cath-D stimulates cancer cell proliferation [95-101] , and increases the metastatic potential [96, 100, 102-104]. Cath-D stimulates cancer cell growth in an autocrine manner [97, 98, 105-107]. Various different mechanisms have been proposed to explain the mitogenicity of cath-D. Intracellular cath-D stimulates high density cancer cell growth by inactivating secreted growth inhibitors, such as heat shock 70 protein [99, 108]. Secreted pro-cath-D may act as a mitogen by competing with IGF2 for interaction with the M6P moieties of the M6P/IGF2 receptor, displacing IGF2 from the IGF1 receptor, and resulting in the activation of the mitogenic IGF1 receptor pathway [109, 110]. Many publications have suggested that the interaction of a part of the cath-D pro-fragment (amino acids 27 to 44) with an unknown cell surface receptor is implicated in its mitogenic function [97, 101, 107, 111-113]. Alternatively, it has also been suggested that the catalytic activity of secreted cath-D may be implicated in releasing growth factors, such as FGF2, from the extracellular matrix [114]. Even though, the extracellular pH in tumors is generally more acidic than that in the corresponding normal tissues [115], the question remains as to whether secreted pro-cath-D could be activated extra-cellularly in a sufficiently acidic milieu. We have demonstrated that a mutated ^D231Ncath-D, which is devoid of proteolytic activity, was still mitogenic for cancer cells both in vitro, in three-dimensional (3D) matrices, and in athymic nude mice [53, 105], suggesting that cath-D can also act by protein-protein interaction [116].

Interactions between stromal and epithelial cells are important in cancer progression and metastasis [117-119]. Stromal and tumor cells can exchange numerous tumor-promoting factors, such as growth factors, cytokines, and proteases. The fibroblast is a major cell type of the stromal compartment and, as such, is intimately involved in orchestrating the stromal side of the dialogue in tissue homeostasis. Cath-D is localized on the surface of breast fibroblasts [6], and can be taken up by fibroblasts [30, 120, 121]. Cath-D has been shown to play a crucial paracrine role in the tumor micro-environment by stimulating fibroblast outgrowth and tumor angiogenesis [53, 122], and possibly by inhibiting anti-tumor responses [123]. More recently, endothelial cells have been shown to secrete pro-cath-D via the action of inflammatory cytokines [124]. A mutant version of cath-D (D231N) that was devoid of catalytic activity, still proved to be mitogenic for fibroblasts, suggesting a mechanism involving protein-protein interaction [120].

Interestingly, some reports have indicated that the cysteine lysosomal cathepsins, cath-L and cath-F, which lack a signal peptide, localize in the nucleus [125, 126].  Nuclear cath-L proteolytically processes CDP/Cux transcription factor [12, 125] and histone H3 [127, 128], and has important functions in the control of cell transformation [129, 130] as well as in differentiation [127]. It has been shown that translation initiation at downstream AUG sites within cath-L mRNA is the first requirement in the chain of events that leads to the presence of active cath-L in the nucleus [125]. There is, at present, no clear evidence for the presence of cath-D in the nucleus, and the nuclear function of cath-D in cancer is still unknown. However, our preliminary experiments strongly suggest the presence of cath-D in the nucleus of cancer cells. This may be due to translation initiation at downstream AUG sites within the cath-D mRNA (Figure 2). Indeed, it is worth noting that, like that of cath-L [125], the coding sequence of cath-D contains several AUG codons that are located downstream of the first AUG codons. Alternatively, two recent reports have suggested that cytosolic mature cath-D may reach the nucleus in apoptosis (Figure 2) [70]. One important question concerns the ability of cath-D to act as a functional enzyme at the neutral pH of the nucleus. Since enzymatic activity by cath-D is achieved at acidic pH, we can reasonably assume that cath-D might be only weakly active in the nuclear milieu.  However, even limited cath-D activity in the nucleus could be compatible with a role in the proteolytic processing of specific nuclear proteins. In contrast, the optimal activity of cathepsins in the acidic environment of the lysosomes is necessary for the terminal degradation of proteins. Thus, the suboptimal pH that prevails in the nucleus should not be taken as an obstacle, but rather as an important element that enables cath-D to play a role in the limited proteolysis of nuclear proteins.  Another possibility is that nuclear cath-D may sequester transcription repressors and/or activators, modulating the composition of the complexes implicated in the tight control of transcription. Our preliminary results indicate that cath-D can indeed interact with a nuclear repressor implicated in cancer. Future studies will clarify whether cath-D participates in the regulation of transcription in cancer by cleaving and/or interacting with nuclear proteins, and thus modulates their activity.