Review

Search for cancer treatment continues: Procathepsin D

¹Aruna Vashishta, ²Vaclav Vetvicka

• ¹Department of Neurological Surgery, University of Louisville, Louisville, KY 40202, USA
• ²Department of Pathology, University of Louisville, Louisville, KY 40202, USA

• Submitted: October 10, 2012
• Accepted: October 19, 2012
• Published: October 19, 2012

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Cathepsin D (CD) has been identified as an aspartic protease involved in protein degradation in the acidic environment of the lysosome, but studies done over the years showed its involvement in other processes like apoptosis, antigen processing, tissue homeostasis suggesting that the enzymatic activity of CD is confined not only to lysosome. Moreover, the enzymatically inactive form of CD, procathepsin D (pCD), has also been shown to be involved in processes like apoptosis and neoplastic growth. Numerous studies done over the past two decades have shown that cancerous cells secrete pCD which acts as a mitogen for both cancerous cells as well as stomal cells by contributing to the processes like angiogenesis, invasion, metastasis and proliferation. In this review, we will discuss the about various forms of CD and their role in different physiological and pathological conditions of cells.

Keywords

Cathepsin D, procathepsin D, cancer, apoptosis, metastasis.

Introduction

Cathepsins are proteases involved in protein degradation in a strong acidic milieu of lysosomes and are subgrouped based on the amino acid present on their active site. Cathepsin D (CD) belongs to group of aspartic proteases and has been shown to be involved in many processes besides general protein turnover, such as activation and degradation of chemokines, polypeptide hormones, growth factors and their receptors [1-4], antigen processing [5-7], tissue homeostasis [8], regulation of apoptosis [9,10], and also in cancer progression and metastasis [11-14]. Since this protein is essential for normal physiological functions as well as implicated in various pathological conditions, to better design the diagnostic as well as therapeutic interventions, it is necessary to understand the feature of the CD involved in these processes.

General Feature of Cathepsin D

Cathepsin D is a soluble lysosomal endopeptidase [EC 3.4.23.5] and its gene is located on chromosome 11p15 and contains 9 exons in humans [15]. It is synthesized on rough endoplasmic reticulum as a pre-proenzyme. After removal of a signal peptide, a 52 kDa procathepsin D (pCD) is glycosylated with two N-linked oligosaccharides modified with mannose-6-phosphate (M6P) residues at asparagines 70 and 199 [16,17] and is transported to Golgi stacks. Glycosylated pCD is then targeted to lysosome via M6P receptors or independently of M6P tag [18,19]. The latter pathway is not fully known but involvement of spingolipid activator precursor protein prosaponin has been reported [20,21]. In the lysosomal compartment, the cleavage of the 44 amino acid N-terminal propeptide results in 48kDa single chain intermediate active enzyme form that undergoes further cleavage into mature two-chain enzyme of a light (14kDa) and heavy (34kDa) chains linked by a non-covalent interactions [22,23]. The role of cysteine proteases, as well as the autocatalytic activity of CD, is involved in pCD/CD processing in lysosomal compartment [24,25]. The human CD catalytic site includes two critical aspartic residues at 33 and 231 that are located on the 14kDa and 34kDa chains, respectively. The presence of pCD/CD is mostly intracellular but its presence as well as activity is reported in extracellular matrix and synovial fluid of cartilage during physiological and pathological conditions [26] and it is also found in human, bovine and rat milk [27-29], serum [30], sweat and urine [30,31] and also secreted by numerous types of cancer [32].

Physiological Functions

To understand the role of CD in organism, the CD knock-out mice were prepared. It was surprising to see that these mice develop normally during embryonic development [33]. This could be due to other cathepsins ensuring the proper protein degradation and turnover during development [34]. However, these mice develop problems two weeks postnatal. CD knock-out mice showed progressive weight loss that is associated with necrosis and hemorrhage of small intestine and increased apoptosis in spleen and thymus. At the terminal stage, these mice develop seizures and become blind due to progressive retinal atrophy and finally die by age of four weeks [8,33]. This suggests that CD is essential for proteolysis of protein regulating cell growth, tissue homeostasis, remodeling and renewal of certain epithelial cells [8].

Unlike mice, the mutation of CD gene in other animals and in humans does not cause visceral and thymus problems but develops a progressive neuronal accumulation of lipofusin (aging pigment) known as neuronal ceroid lipofucinosis (NCL) characterized by neurodegeneration, developmental regression, visual loss and epilepsy [35,36]. The severity in the CD-derived form of NCL depends on residual CD enzymatic activity. The exact mechanism of neuron death is not clear but studies suggest that CD activity is important in postnatal tissue homeostasis that includes tissue renewal, regulation of aging and apoptosis.

The role of CD in apoptosis remains controversial. It was described in knock-out mice as well as in different models over the years [34]. Both the pro-apoptotic as well as anti-apoptotic activity of pCD/CD has been shown. Some studies suggested that partial permeabilization of lysosomal membrane caused release of cathepsins and other proteases into cytosol subsequently triggering the apoptotic machinery leading to apoptosis [37,38]. On the other hand, massive disruption of lysosomal membrane leads to necrosis [39]. The direct role of CD in apoptosis was shown in staurosporine-induced apoptosis of T-lymphocytes where it triggers Bax activation and relocation to the mitochondria that activates apoptosis-inducing factor [40]. In another study of TNF-mediated apoptosis, CD has been shown to be involved in cleavage of BCl2 family member Bid that releases cytochrome c from mitochondria and activates caspases 9 and 3 [9]. Many studies also suggested that CD is a mediator of apoptosis induced by various cytotoxic and stress stimuli such as oxidative stress [41,42], serum starvation [43], chemotherapeutic-agents [44,45], interferon-gamma [46] and resveratrol [47]. The involvement of CD catalytic activity in the process of apoptosis is questionable as CD is optimally active at acidic pH and cytosol is, for the most part, only slightly acidic. Moreover, most of the studies used pepstatin A to block the catalytic activity of CD, which is a potent but unspecific inhibitor since it inhibits other proteases [48]. Also, the microinjection of wild-type as well as catalytic inactive mutant of CD enhanced apoptosis in human fibroblast [49]. In another study, the apoptotic response to chemotherapy enhanced when wild-type or mutant CD was exogenously expressed in rat tumor, suggesting that CD might interact with some member(s) of apoptotic pathway [50]. Conversely, the study done with xenografts of cancer cells overexpressing CD showed less apoptosis than mock-transfected cancer cells [13] and transfection of wild-type or catalytic mutant form of CD promotes survival of CD-deficient fibroblasts [51], suggesting the anti-apoptotic role of CD.

Altered expression of CD has also been reported in the skin during wound healing, epidermal differentiation and pathological conditions such as psoriasis [52,53]. Protein level as well as activity of CD increases during epidermal differentiation and inhibiting CD activity by pepstatin A significantly delay permeability barrier repair after experimental disruption. It is reported that CD could be one of the proteases involved in intracellular activation of transglutaminase I into enzymatically active form [54] that is involved in crosslinking of the proteins of cornified envelope of skin.

In addition to the role of CD enzymatic activity in epidermal development, we observed that the keratinocyte cell line HaCaT showed increased proliferation and regeneration in the presence of enzymatically inactive pCD. We also observed that these cells substantially increase secretion of pCD under conditions like oxidative stress [55], suggesting that the inactive form of pCD might play a role in the regeneration of skin.

Role of cathepsin D in pathological conditions

The implication of pCD/CD in various pathological conditions has been shown over the years. The role of this protease in diseases like atherosclerosis [56,57], Alzheimer’s disease [58,59], Parkinson’s disease [60,61], psoriasis [52,53] is indicated, but most of the studies focused on its role in cancer.

Extensive studies done over the past two decades have documented the pCD/CD overexpression and hyper-secretion in several types of cancer (breast cancer, ovarian cancer, head and neck cancer, bladder cancer, colorectal cancer, neuroblastoma, prostate cancer, lung cancer, and melanoma). Clinical studies conducted in 1990’s revealed that the CD levels in primary breast cancer cytosols is an independent prognostic parameter that correlates with the incidence of metastasis and short survival [62,63]. Moreover, a meta-analysis of studies on node-negative breast cancer [64] and Rotterdam study of 2810 patients [65] showed that the higher levels of CD to be a marker of aggressiveness. Using the monoclonal antibodies specific for the pro-form, it has been shown that the pCD levels increase in plasma of patients with metastatic breast carcinoma [66]. Various approaches, such as immuno-histochemistry, cytosolic immunoassay, in situ hybridization, Northern and Western blot analyses to detect the CD levels in neoplastic tissues, revealed that in a majority of cancers pCD is overexpressed 2-to-50 fold compared to the control tissue.

CD synthesis is controlled by progesterone, estrogen and certain growth factors (e.g., IGF1,EGF). Progesterone and its derivatives increase the rate of uterine CD synthesis [67] and in estrogen receptor (ER) positive breast cancer cell lines by estrogen [68]. In ER positive breast cancer cell lines, estrogen and growth factors stimulate CD protein synthesis and mRNA accumulation [68] by direct interaction at the promoter site by estrogen responsive elements [69]. However, in case of ER negative breast cancer cell line the expression and secretion of pCD is constitutive [70].

That procathepsin D acts as mitogen for cancer cell was first shown by Vignon and colleagues when they demonstrated that the purified pCD from MCF-7 breast cancer cells stimulated growth of these cells [71]. Studies conducted in our laboratory and that of others have shown that pCD secreted from cancer cells serves as autocrine growth factor for breast [72], prostate [73], ovarian [74] and lung cancer cells [75]. In addition, HBL-100 human breast epithelial cell line transfected with human pCD cDNA increased metastatic potential of this cell line both in vivo and in vitro [76]. Another independent study has shown similar results by transfection of human pCD cDNA in rat tumor cells with increased proliferation, invasion and metastasis in vitro and in vivo [13]. Further experiments using either the antisense gene transfer [77], ribozymes [78], or RNA interference [79] to downregulate the pCD expression in the breast cancer cell line MDA-MB-231, showed significant reduction in their metastatic potential. Also, tumor growth was inhibited by anti-pCD antibodies both in vitro and in vivo [72,73,80].

Over the years, many mechanisms have been proposed that are responsible for the mitogenicity of CD. Earlier studies suggested that the enzymatic activity of CD is involved: several growth factors, growth factor receptors and extracellular matrix components have been shown to be among the CD substrate. In addition, the moderate acidic environment of tumors [81,82] could convert the inactive pCD to the active enzyme. However, we showed that the mitogenic activity of pCD is not inhibited by pepstatin A [72,73]. Moreover, we and others have shown that enzymatically inactive pCD mutants stimulate growth of cancer cells in vitro and in vivo in a similar manner as wild-type pCD [76,83]. The other mechanism proposed for the mitogenic effect of pCD is by binding to the surface of breast cancer cells [84,85] and the only receptor to which pCD/CD binds is the M6P receptors that recognizes the M6P tag on glycoproteins. Different studies showed that neither binding nor pCD growth-promoting potential is blocked by M6P, anti-M6P receptors or pCD deglycosylation [72,83,85,86]. Further, we showed that mutation in one or both glycosylation sites of pCD only slightly lowers the pCD mitogenic and invasive properties in vitro and in vivo [76]. The binding of pCD to M6P/IGF-II receptor may decrease its binding to more natural ligands like IGF-II and thus affect their biological functions [87].

The work done in our laboratory found that binding to cancer cells as well as the mitogenic activity of pCD is blocked by antibodies specific for propeptide part of pCD, also known as activation peptide (AP) [72,73,80]. Using synthetic peptides corresponding to different parts of AP, we demonstrated that the region spanning 33-44 aa is responsible for binding of pCD to cancer cell [80,85]. We also showed using synthetic AP, anti-AP antibodies or mutant pCD with deleted AP, that the AP alone is sufficient to stimulate growth of breast, prostate and lung cancer cells in vitro and in vivo [72,73,75,76,80,86]. Bazzet et al. also showed the mitogenic effect of AP on ovarian cancer cells [74], but Glondu et al. could not confirm the growth-promoting effect of AP in their experimental conditions [83].

The hyper-secreted pCD not only has autocrine effect on cancer cells but also has paracrine action on fibroblasts. The interaction between the stromal cells and cancer cells is very essential for neoplasis [88]. Laurent-Matha et al. have shown that the pCD is required for invasive growth of fibroblast using 3D co-culture assay with cancer cells either secreting or not secreting pCD. In addition, they showed that ectopic expression of pCD in CD-deficient fibroblasts is associated with fibroblast proliferation, survival, motility and invasive capacity by activation of the ras-MAP kinase pathway [53]. Recently, the same group identified the low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) as a fibroblastic cell surface receptor that interact with pCD and is essential for outgrowth of fibroblasts [89].

Tumor growth depends on angiogenesis and correlation of stromal CD expression with microvessel density has been shown in ovarian cancer [90] as well as in breast cancer tumors [91]. The immunohistochemical study has shown that CD stimulates not only cancer cell growth but also promotes tumor angiogenesis and, further, that this action is independent of its enzymatic activity [51]. The overexpression of CD by cancer cells significantly stimulated tumor angiogenesis in tumor xenografts in athymic mice [51]. The mechanism by which CD promotes angiogenesis is not fully defined but some studies suggested that CD may influence the production and degradation of both activators and inhibitors of angiogenesis. Briozzo et al. observed that CD facilitates the release of pro-angiogenic bFGF from ECM [92]. Moreover, it has been described in vitro that CD is able to activate/inactivate cryptic anti-angiogenic factors including angiostatin, 16K prolactin and endostatin [93-95]. We also observed that several regulators of angiogenesis are differentially expressed upon treatment of breast cancer cells with AP [96].

Taken together, the proposed model of pCD action is that over-expressed pCD escapes the normal targeting pathways and is hyper-secreted by cancer cells and exerts its autocine and paracrine effect on epithelial cells and stromal cells, respectively, by binding to cell surface receptor. This interaction activates the signal transduction pathway (like MAPK pathway) that results in differential expression of cancer promoting genes [96] including various cytokines [97,98].

Conclusions

In addition to its primary function as the lysosomal aspartic proteinase, CD has been shown to be involved in other physiological functions such as apoptosis and tissue homeostasis. The enzymatically inactive form pCD has been shown to be secreted by various types of cells like macrophages, mammary epithelial cells and different types of cancerous cells. The secreted pCD acts as mitogen in autocrine and paracrine manner and different studies have shown that this process is independent of its enzymatic activity, suggesting that pCD/CD interacts with other molecules and thus influences cell signaling. The better understanding of these molecules and pathways will help in designing of diagnostic and therapeutic interventions.

Authors contribution

Both authors contributed equally.

Conflict of interests

Authors declare that there is no conflict of interests.

Funding

None

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