Intracellular signaling by cathepsin X: Molecular mechanisms and diagnostic and therapeutic opportunities in cancer

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Contents lists available at ScienceDirect

Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer

Review

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Intracellular signaling by cathepsin X: Molecular mechanisms and diagnostic and therapeutic opportunities in cancer

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Janko Kos a,b,∗ , Tjaˇsa Viˇzin a , Urˇsa Peˇcar Fonovic´ a , Anja Piˇslar a a

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b

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Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia Department of Biotechnology, Joˇzef Stefan Institute, Ljubljana, Slovenia

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a r t i c l e

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i n f o

a b s t r a c t

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Keywords: Cathepsin X Proteolysis Cancer Adhesion Migration Signaling

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1. Introduction

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Cathepsin X is a cysteine carboxypeptidase, localized predominantly in immune cells, regulating their proliferation, maturation, migration and adhesion. It has recently been confirmed as a significant promoter of malignant progression. Its role in signal transduction was first implied through the interaction with integrin receptors, either by binding with the RGD motif or by proteolytic cleavage of the C-terminal amino acids of the cytosolic part of the integrin beta chain. Several other molecules, involved in cellular signaling, have since been shown to be targets for cathepsin X, such as ␥-enolase, chemokine CXCL-12, bradykinin, kallidin, huntingtin and profilin 1. In cancer, cathepsin X regulates adhesion of tumor and endothelial cells and their migration and invasion through the extracellular matrix. It also promotes tumor progression by bypassing cellular senescence and by inducing an epithelial–mesenchymal transition. The high RNA and protein levels of cathepsin X, found in tumor samples and bodily fluids of patients with various cancer types, further support its active role in tumor progression. Its prognostic value and relation to response to chemotherapy confirm cathepsin X as a new target for improving diagnosis and treating cancer patients. © 2014 Published by Elsevier Ltd.

Peptidases are enzymes that catalyze the hydrolysis of peptide bonds [1,2]. They are involved in practically all physiological processes and failure in their expression, activity or localization can lead to pathological processes and diseases like rheumatoid arthritis, osteoarthritis, cancer, neurodegenerative and cardiovascular diseases, viral infections, atherosclerosis, periodontitis and osteoporosis [3]. In cancer, peptidases are in the first line in degrading proteins of extracellular matrix (ECM) and basal membrane, a prerequisite for tumor invasion, angiogenesis and metastasis. ECM is a complex structure composed of various proteins, including collagens and fibrillar glycoproteins that, under normal conditions, constitute a barrier for cells. A number of peptidases are involved in the degradation of ECM, including cysteine peptidases cathepsins B and L, aspartic peptidase cathepsin D, and metallo- and serine peptidases, which all act either directly or by activating other peptidases

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and proteins involved in the proteolytic cascade [4,5]. Besides their direct action on the ECM, some peptidases release growth factors bound to proteins of the ECM or activate cytokines, both stimulating proliferation of tumor and endothelial cells [6]. Moreover, they can potentiate malignant progression by inactivating endogenous peptidase inhibitors [7,8]. Peptidases also regulate the apoptosis of tumor cells [9], impair the anti-tumor immune response and enhance immune tolerance [10]. Peptidases are also involved in intracellular signaling, resulting in the transformation and differentiation of tumor cells, in cell adhesion and cytoskeleton remodeling and in modulating growth and development of cancer stem cells and in the latters’ transition between epithelial and mesenchymal types [11]. In this paper we review the properties, molecular mechanisms and function of cathepsin X (also named cathepsin Z or P), a cysteine peptidase that is actively involved in cell signaling, promoting adhesion, migration and invasion of tumor cells. Additionally, we discuss the opportunities this new target provides toward better diagnosis and treatment of cancer patients. 2. Cysteine cathepsins

Q2

∗ Corresponding author at: Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia. Tel.: +386 40792639; fax: +386 1722315. E-mail address: [email protected] (J. Kos).

561 genes encoding peptidases have been identified in the human genome. Of these, 148 encode cysteine peptidases, including a group of 11 lysosomal cysteine peptidases, the cathepsins [1].

http://dx.doi.org/10.1016/j.semcancer.2014.05.001 1044-579X/© 2014 Published by Elsevier Ltd.

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The latter exhibit different expression patterns, levels and specificities, all of which contribute to their different physiological roles. Some of them, like cathepsins B, H, L and C, are present in all tissues, whereas others (cathepsins S, V, X, O, K, F and W) are expressed only by specific cell types. Cysteine cathepsins cleave peptide bonds predominantly as endopeptidases, with the exception of cathepsins B, C, H and X [1,12]. The latter act as exopeptidases due to additional structural elements in their active site clefts which both prevent binding of extended substrates into the active site cleft and facilitate binding of either the C- or N-terminal end of substrates, thus conferring on the enzyme carboxy- or aminopeptidase activity [13]. Based on their specific structures cathepsins B and X cleave their substrates as dipeptidylcarboxypeptidase and as carboxymonopeptidase, respectively [14], while cathepsins H and C cleave their substrates as aminopeptidases [15]. However, cathepsins B and H exhibit endopeptidase, as well as exopeptidase activity [16]. Cysteine cathepsins were long believed to be responsible for terminal protein degradation in the lysosomes, however, they have been found more recently to play, in addition, more specific roles in a number of other important cellular processes and pathologies [17]. Recent studies have also implicated cysteine cathepsins in processes occurring outside lysosomes and endosomes, e.g. in the nucleus, the cytosol and on the cell membrane, and they have been shown even to be secreted into the extracellular environment. Localization of cysteine cathepsins outside lysosomes has been associated with physiological processes of pro-hormone activation, apoptosis and cell migration, while the extracellular role of cathepsins has been studied most intensively in processes of cancer progression [4,18].

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3. Cathepsin X

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The gene for cathepsin X (EC 3.4.18.1), its structure and activity show several unique features that distinguish it from other human cysteine cathepsins. It has a very short pro-region and a three residue insertion motif which forms a characteristic “mini loop”. Cathepsin X exhibits solely carboxypeptidase activity and is activated by other lysosomal endopeptidases such as cathepsin L [19]. Contrary to first reports, cathepsin X is not widely expressed in cells and tissues, but is restricted to cells of the immune system, predominantly monocytes, macrophages, microglia and dendritic cells [20,21]. It regulates the proliferation, maturation, migration and adhesion of immune cells, as well as their phagocytosis and signal transduction [22]. Higher levels of cathepsin X are also associated with various diseases such as cancer [11,23–26], inflammatory diseases [27] and inflammation related neurodegenerative disorders [21,28], Helicobacter pylori infection [25,29], induction and maintenance of chronic pain [30] and tuberculosis [31]. Recently several molecular targets of cathepsin X exopeptidase activity have been identified, including the ␤-chain of integrin receptors [32–34], ␥enolase [35], chemokine CXCL-12 [36], bradykinin and kallidin [37], huntingtin [38] and profilin 1 [39]. 3.1. Cathepsin X promotes signal transduction by interaction with integrin receptors The involvement of cathepsin X in signal transduction was first implied by the motifs present in its pro-form (RGD: Arg–Gly–Asp) and its mature form (ECD: Glu–Cys–Asp) [40,41] that bind to integrin receptors – heterodimeric membrane glycoproteins that participate in signal transduction into and out of cells. Cathepsin X was also shown to bind to cell surface heparan sulfate proteoglycans, which regulate cathepsin X cellular trafficking and enzymatic activity [42] and are partners of integrins in focal adhesion formation, playing an important role in cell adhesion and signaling.

Fig. 1. Co-localization of cathepsin X and profilin 1 in human prostate cancer cells PC3. Proteins were visualized by immunofluorescence staining using primary antibodies to cathepsin X and profilin 1 followed by Alexa Fluor 488 and 555 conjugated secondary antibodies. Profilin 1 (red) and cathepsin X (green) staining showed a strong co-localization in the perinuclear region and at the plasma membrane. Fluorescence microscopy was performed by Carl Zeiss LSM 710 confocal microscope with ZEN 2011 image software. Bar represents 10 ␮m. The image was prepared by authors and has not been published elsewhere.

Procathepsin X interacts with ␣v␤3 integrin through the RGD motif in lamellipodia of human umbilical vein endothelial cells [43], mediating cell adhesion properties. Strong co-localization of procathepsin X with ␤3 integrin subunit was also demonstrated in pro-monocytic U-937 cells [34]. Procathepsin X also binds another RGD binding integrin, ␣v␤5, present on plasma membrane of pancreatic ductal adenocarcimoma cells [44]. Again, cathepsin X was shown to mediate the adhesion of these cells and interestingly, its expression was negatively regulated by S100PBP, a binding partner of the S100 family of calcium binding proteins, frequently upregulated in pancreatic cancer. Further to the interaction of procathepsin X with the RGD motif in a proregion, active mature cathepsin X processed from inactive precursor by cathepsin L also interacts with integrin receptors. Active cathepsin X was co-localized specifically with the ␤2 integrin receptors that are present predominantly in cells of monocyte/macrophage lineage [34]. The interaction of cathepsin X with ␤2 integrin subunit was confirmed by immunoprecipitation and fluorescence resonance energy transfer [32]. Cathepsin X was also found to cleave sequentially four C-terminal amino acids of ␤2 integrin subunit until reaching a proline in the penultimate position, confirming a previous observation that proline in the S2 position impairs cathepsin X proteolysis [45]. In macrophages and dendritic cells this cleavage results in activation of the ␤2 integrin receptor Mac-1 (CD11b/CD18), which is associated with increased cell adhesion, phagocytosis and T lymphocyte activation. Inhibitors and monoclonal antibody capable of impairing cathepsin X enzymatic activity, reduced Mac-1 dependent binding of differentiated U-937 cells to fibrinogen and to a polystyrene surface. The co-localization of active cathepsin X with ␤2 integrin chain was particularly enhanced in interactions of monocyte/macrophages with endothelial and tumor cells [34] Q4 (Figs. 1 and 2). In addition to its role in monocytes and macrophages the Mac-1 receptor is crucial for the functioning of dendritic cells (DC) that are responsible for effective antigen presentation and initiation of a T cell-dependent immune response. Maturation of dendritic cells is accompanied by a range of morphological and cytoskeleton structure changes. In response to maturation stimuli, DCs rapidly adhere, develop the polarity, and modulate cytoskeleton [46]. The adhesion of immature DCs to the ECM is accompanied by

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Fig. 2. Involvement of cathepsin X in signal transduction. Procathepsin X interacts with ␣v␤3 integrin through RGD motif mediating cell adhesive properties (A). Cathepsin L is proposed to activate procathepsin X into the lysosomal vesicles and active cathepsin X interacts with ␤2 integrin receptor (B), profilin-1 (C) and ␥-enolase (D) where active cathepsin X sequentially cleaves the C-terminal amino acids of the proteins. The image was prepared by authors and has not been published elsewhere.

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recruitment of Mac-1 integrin receptor. During maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac-1 activation and, consequently, cell adhesion [47]. In mature DCs, cathepsin X is redistributed from the plasma membrane back to the perinuclear region, which coincides with the detachment of DCs and acquisition of the mature phenotype. In T cells, cathepsin X regulates the function of another integrin receptor – lymphocyte function associated antigen-1 (LFA-1) – by cleaving C-terminal amino acids at its ␤2 chain. LFA-1 is one of the key regulators of physiological T cell functions, including migration and formation of the immunological synapse by binding intracellular adhesion molecule-1 (ICAM-1). LFA-1 can act as a true signaling receptor, causing the reorganization of F-actin that leads to cytoskeletal changes of the cell [48] and a switch from a spherical to a polarized shape. The interaction of cathepsin X with LFA-1 promotes cytoskeleton-dependent morphological changes and migration across 2D and 3D models of ICAM-1 and Matrigel [33]. Gradual cleavage of the ␤2 cytoplasmic tail of LFA-1 modulates the affinity of LFA-1 for structural adaptors talin-1 and ␣-actinin-1, enabling a stepwise transition between intermediate and highaffinity conformations of LFA-1 [49]. Conformational changes are of vital importance for the regulation of LFA-1 affinity and the binding of ICAM-1. LFA-1 fine-tuning by cathepsin X enables the trafficking of T cells which is accompanied by extensive actin remodeling via selective binding of actin proteins talin-1 and ␣-actinin-1. The interaction between cathepsin X and LFA-1 is particularly evident at the trailing edge protrusion, the uropod, which plays an important role in T lymphocyte migration and cell-cell interactions [33]. Uropods of T lymphocytes with upregulated cathepsin X enlongate to extreme lengths to form cell-to-cell connections,

the nanotubes [50]. Membrane nanotubes provide a new principle of cell–cell communication, enabling transmission of complex and specific messages to distant cells through a physically connected network. 3.2. Cathepsin X processes -enolase, polyQ peptides and huntingtin, all involved in aging and neurodegenerative processes Besides immune cells the brain has been reported to be rich in cathepsin X. It is localized mainly in cells of glia, but also in neurons, oligodendrites and ependymal cells. The expression and activity of cathepsin X are increased in cells that are in the phase of aging or are subjected to degenerative processes [28] (Table 1). The activation of microglia is a typical histopathological marker of neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) [51]. The activation of microglia was for a long time seen as a secondary event following neuron damage. Numerous studies now show that activation of microglia contributes to the processes of neurodegeneration by releasing neurotrophic factors that act as neuroprotective or proinflammatory and/or cytotoxic factors that contribute to the degeneration of neurons [52]. Activated microglia also release lysosomal cysteine peptidases, including cathepsin X [53]. Neurotrophic factors regulate the survival, differentiation and regeneration of neurons and their connections. They bind to specific receptors on the neuron membrane, in this way activating two main kinase dependent signaling pathways, important for the survival of neurons, i.e. MAPK/ERK and PI3K/Akt. The role of neurotrophic factors is especially important in neurodegenerative diseases since

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4 Table 1 Molecular targets of cathepsin X. Target

Cathepsin X action

Function

Reference

␣v␤3 and ␣v␤5 integrin receptors

Binding through RGD motif in propeptide

[43,44]

Heparan sulfate proteoglycans ␤2 integrin receptors Mac-1 and LFA-1

Binding to cell surface heparan sulfate proteoglycans Cleavage of four C-terminal amino acids

Cell adhesion and migration Angiogenesis Metastasis Cell adhesion and signaling

[32–34,47,49,50]

Profilin 1

Cleavage of up to five C-terminal amino acids

␥-Enolase

Cleavage of two C-terminal amino acids

CXCL-12 chemokine

Cleavage of up to fifteen C-terminal amino acids Cleavage of one C-terminal amino acid from bradykinin and kallidin Cleavage of two C-terminal amino acids Cleavage of huntigtin C-terminal cleavage of shorter peptides, generated by cathepsin L

Cell adhesion and migration Phagocytosis T lymphocyte activation Maturation of dendritic cells Immunological synapse formation Cell-cell communication Actin polymerization Cell migration, invasion and adhesion Clathrin binding Regulation of neuron survival, proliferation and neurite outgrowth Impaired adhesion and migration of hematopoietic stem and progenitor cells Modulation of the kallikrein–kinin system Conversion of angiotensin I to angiotensin II Increased cell toxicity in Huntington’s disease Decreased cell toxicity in neurodegenerative diseases

[37] [38] [63]

Bradykinin, kallidin Angiotensin I Huntingtin PolyQ peptides

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they may protect affected neurons from destruction or trigger their re-growth and recover their function. ␥-Enolase, a cytoplasmic enzyme involved in glycolysis, is an important neurotrophic factor [54]. It is a dimer composed of non-covalently linked subunits ␣, ␤ and ␥. Iso-enzyme ␥␥, i.e. ␥-enolase or neuron-specific enolase (including also ␣␥ isoenzyme), is present in high concentrations in neurons and peripheral endocrinal cells in brain tumors. The C-terminal end of ␥-enolase is responsible for the neurotrophic function. Other enolase isoforms do not exhibit neurotrophic activity, despite having similar amino acid sequences at the C-terminal end. It was recently shown that cathepsin X cleaves two amino acid residues at the Cterminal end of ␥-enolase and that the truncated form no longer exhibits neurotrophic activity [35]. Additionally, ␥-enolase exhibits a neurotrophic function only if it binds via the PDZ motif to ␥1-syntrophin, a scaffold protein that translocates ␥-enolase to the plasma membrane [55]. Cleavage of the C-terminal end of ␥-enolase disrupts the PDZ binding motif, prevents binding to ␥1-syntrophin and, consequently, abolishes neurotrophic function. ␥-enolase, translocated toward the plasma membrane, enhances MAPK/ERK and PI3K/Akt signaling pathways, normally triggered by the activation of Trk receptor [56]. In brains of Tg2576 mice overexpressing amyloid precursor protein, used as a model of Alzheimer disease, ␥-enolase and cathepsin X are expressed abundantly around amyloid plaques. The C-terminally cleaved form of ␥-enolase is present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. In the cell model using mouse microglial cell line EOC 13.31 and primary microglia, ␥-enolase was shown to exert a neuroprotective role against amyloid-␤ peptide toxicity that was reversed by cathepsin X activity [57]. ␥-Enolase, whose overexpression is typical in neurogenic and neuroendocrine tumors [58], was proposed as a tumor marker in small cell lung cancer, neuroblastoma, melanoma and seminoma. Its high concentration was related to the increased aerobic glycolysis and thus proliferation of cancer cells [59]. The prevalence of aerobic glycolysis over oxidative phosphorylation is a phenotype of a great variety of solid tumors. Tumor cells displaying this phenotype, also referred to as the Warburg effect, carry out aerobic glycolysis even at the sufficient supply of oxygen. Enolase catalyzes

[42]

[39]

[35] [36] [37]

the conversion of 2-phosphoglycerate to phosphoenolpyruvate, the penultimate step in glycolysis, a process converting glucose into pyruvate with concomitant formation of high-energy compounds ATP and NADH [60,61]. ␥-Enolase was suggested to be involved in malignant processes also as a pro-survival factor [62], as had been demonstrated for neuronal cells, however, the role of both intact and C-terminally cleaved forms remains to be elucidated. Besides processing ␥-enolase, cathepsin X was reported to digest aggregation-prone peptides with polyglutamine stretches as well as huntingtin, both exhibiting toxicity when accumulating in neuronal cells [38,63]. However, in the case of huntingtin, the resulting peptides were reported to increase toxicity in neuronal cells [38] while C-terminal cleavage of shorter peptides, generated from polyQ peptides by cathepsin L was reported to decrease toxicity [63].

4. Tumor promoter functions of cathepsin X Dating from the discovery of cathepsin X, evidence has increased for its involvement in malignant processes. The first hint was the location of the gene encoding cathepsin X in chromosomal region 20q13, which is frequently amplified in several cancer types [64]. It is believed that this region may contain one or more oncogenes and cathepsin X was suggested as a possible candidate. The protein levels of cathepsin X were found to be increased in several cancer types, first in gastric [25] and prostate cancer [23]. Immunohistochemical analyses demonstrated strong staining for cathepsin X in prostatic intraepithelial neoplasias and invasive adenocarcinomas, and it was suggested that cathepsin X plays a role in the early tumorigenesis of prostate cancer. In gastric cancer cathepsin X staining was significantly more common and intense in intestinal type gastric cancer than in the diffuse type [25]. Increased cathepsin X expression was detected in H. pylori-infected gastric mucosa [65] and, moreover, its location and activity in monocytes treated with H. pylori antigens was shown to predict the effectiveness of H. pylori eradication [29]. In lung tumors, strong staining of cathepsin X was observed in infiltrated immune cells, whereas in tumor cells it was very weak [20]. RNA and protein levels of cathepsin X in hepatocellular carcinomas were found to be higher than in non-tumorous liver tissue [11]. Overexpression of the cathepsin X

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gene was associated with the aggressive phenotype of malignant melanoma [24]. 4.1. Cathepsin X levels in tumors and body fluids from cancer patients

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The correlation of cathepsin X levels in tumors and body fluids of cancer patients with clinical and pathological parameters further supports its active role in malignant progression. In initial clinical studies, the levels of the active form of cathepsin X were evaluated in extracellular fluids of healthy persons and of patients with breast and ovarian tumors. In general, the levels of active enzyme in samples from healthy persons were very low, as shown by immunoassay (ELISA) [20]. Levels in sera from patients with breast cancer were moderately higher [66]. A subgroup of inflammatory breast cancer patients exhibited significantly lower levels of cathepsin X than those with non-inflammatory breast cancer. Active cathepsin X was present in higher concentrations in epithelial ovarian cancer cyst fluid, but its levels did not correlate with any clinical or pathological parameter [67]. It became apparent that the extracellular levels of active cathepsin X are too low and therefore not able to be of clinical relevance. However, Wang et al. reported that cathepsin X mRNA expression is higher in hepatocellular carcinomas than in healthy control liver tissue and that it correlates strongly with advanced clinical stage and shorter overall survival [11]. Further, Zhang et al. [68] measured cathepsin X serum levels in patients with lung cancer and found that high levels correlated significantly with shorter overall survival. The immunoassay used in the latter study presumably detects also procathepsin X, which is secreted from tumor and immune cells in large amounts (Viˇzin, unpublished results). The results are consistent with the study of Nagler et al. [27] who measured plasma levels of total cathepsin X (both the pro- and active forms). They were significantly elevated in patients who experienced severe trauma, particularly in those who died during the post-traumatic period. Levels of total cathepsin X in extracellular fluids may thus reflect the tumor and patient status better than the levels of active cathepsin X. Studies on patients with colorectal cancer and matching patients with adenomas, non-neoplastic findings and healthy persons confirm this conclusion. Significant differences in total cathepsin X levels between the groups could not be demonstrated. However, within the group of patients with colorectal cancer, higher levels of total cathepsin X correlated significantly with shorter overall survival, showing the potential prognostic value of serum total cathepsin X [26]. These results were confirmed on a larger cohort of patients with colorectal cancer and the correlation with the applied chemotherapy suggests total serum cathepsin X to have predictive value [69].

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4.2. Cathepsin X deficient animal tumor models

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The results obtained from mice deficient in expression of cathepsins suggested that the function of cathepsin X in tumor progression is redundant to that of the related carboxypeptidase cathepsin B, a validated target in cancer. In a mouse model of transgenic polyoma middle T oncogene (PymT)-induced breast cancer deficient for cathepsin B, tumor growth and metastasis were lower, but only to a certain stage, compared to wild type mice. Interestingly, a higher expression of cathepsin X was observed in cathepsin B deficient PymT mammary tumor cells [70] and it appeared that it could compensate for the lower invasive potential caused by lack of cathepsin B. Mice with cathepsin X deficiency also revealed a longer tumor-free period for breast cancer, and delayed tumor growth and metastasis as compared to the wild type mice [71]. Nevertheless, in mice with double deficiency (for both cathepsins B and X) tumor growth and metastasis formation were significantly

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lower than that in mice with single deficiency or in wild type, showing the additive effect of the two cathepsins. This further supports their redundant function in tumor progression. Similarly, in mice with double deficiency, laser-induced choroidal neovascularisation was significantly reduced whereas in single knockouts it was not altered [72]. This also supports a redundant function for the two cathepsins in tumor angiogenesis and suggests that only inhibition of both enzymes can reduce the angiomodulatory potential of tumor cells. 4.3. Cathepsin X mediates adhesion and migration of tumor cells through interactions with integrin receptors and profilin 1 However, though the functions of cathepsins B and X are redundant in processes of tumor progression, the molecular mechanisms they utilize in these processes cannot be the same. For cathepsin B it is well established that it promotes tumor growth, invasion, metastasis and angiogenesis by extensive degradation of ECM, acting as an endopeptidase. ECM provides a physical barrier and its breakdown may create a pathway for tumor and endothelial cells to migrate to distant sites. On the other hand, cathepsin X acts solely as a carboxypeptidase and is not capable of degrading ECM. One of the possible mechanisms directing its action toward tumor progression is the modulation of the adhesive properties of tumor cells by interaction with integrin receptors. Procathepsin X, localized on plasma membrane or secreted from tumor and infiltrated immune cells, was shown to bind via the RGD motif ␣v␤3 and ␣v␤5 integrin receptors, which are commonly seen in cancer cells and have been associated with tumor growth, angiogenesis and metastasis. In the case of endothelial (HUVEC) and pancreatic cancer cells (PDA), cathepsin X/integrin binding significantly changed cell adhesion to the proteins of ECM [43,44]. Although an exact mechanism was not proposed, this change could affect the migration of tumor cells through the ECM and the adhesion of circulating tumor cells to microvascular endothelium at distant sites. The adhesion of cancer cells can also be regulated by active cathepsin X modulating integrin receptors by proteolytic cleavage. Procathepsin X is processed to active enzyme by cathepsin L, an endopeptidase known to be overexpressed in the most of tumor types [4]. As noted above, active cathepsin X is able to cleave up to four amino acids at C terminus of ␤2 chain of integrin receptors. The cleavage stabilizes the active conformation of ␤2 integrin extracellular domains and enhances the binding of the components of ECM and other ligands. Although the concentration of ␤2 integrins in tumor cells is not as high as those of its ␤3 or ␤5 counterparts, this mechanism could be important and the interplay between active and procathepsin X may regulate the adhesion and detachment of tumor cells to ECM and the interaction with infiltrated immune cells. An intriguing possibility is that cathepsin X may switch the migration mode of tumor cells from mesenchymal-like, typical of the most tumor cell phenotypes, to ameboid-like, typical of T cells. Overexpression of cathepsin X in T cells greatly enhances their migration through the ECM, due to permanent activation of ␤2 integrin receptor LFA-1. The compensation of cathepsin B, which is a key player in mesenchymal-like invasion and migration, with higher expression of cathepsin X in tumor cells, deficient in cathepsin B [70], supports this idea. Tumor cells without cathepsin B are still able to invade and migrate, either by degrading ECM by other endopeptidases or by a switch in the migration mode. The latter deserves further investigation to define the contribution of particular peptidases and their interplay in different migration modes. Profilin 1 is the most recently identified target for cathepsin X carboxypeptidase activity [39]. It is a known tumor suppressor, downregulated in various types of cancer, such as breast cancer [73,74], hepatocarcinoma [75], bladder cancer [76], pancreatic

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cancer [77] and in radiation resistant head and neck squamous cell carcinoma cells [78,79]. Furthermore, the level of profilin 1 expression may be a predictor of malignant tumor aggressiveness, response to antitumor therapy and risk of recurrence [79]. In general, higher expression of profilin 1 correlates with lower motility of the cells [80]. Its function is associated with actin polymerization [81] that drives the formation of membrane protrusions. In this process its interaction with actin and poly-l-proline ligands is essential. Alternatively, in the actin-independent pathway, profilin 1 controls cell motility by membrane recruitment of lamellipodin through interaction with phosphatidylinositol lipids (mechanisms reviewed by Ding et al. [82]). Since the binding site for poly-lproline ligands and, partly, for phosphatidylinositol lipids is located at the C-terminus of profilin 1 [83], cleavage by cathepsin X could have an important impact on its physiological function. In prostate cancer PC-3 cells, either RNA silencing or inhibition of cathepsin X by epoxysuccinyl inhibitor AMS-36 significantly decreased cell migration, invasion and adhesion, together with enhanced actin polymerization and binding of profilin 1 with clathrin, a known poly-l-proline binding partner involved in endocytosis [39]. Since migration, actin polymerization and endocytosis are essential tumorigenic processes, profilin 1 could be central to cathepsin X action in tumor cells. Further, the results suggest the possible application of cathepsin X inhibitors in the treatment of malignant diseases.

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4.4. Bypassing cellular senescence

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Another explanation for the compensatory role of cathepsin X in tumor progression is the bypassing of cellular senescence, a tumor suppressor mechanism that prevents the proliferation and invasion of malignant cells [84]. Cathepsin X deficient murine embryonic fibroblasts and neonatal human dermal fibroblasts underwent accelerated cellular senescence, resulting in a reduced proliferation rate and increased expression of senescence-associated genes such as p16, p21, p53, and caveolin, typical markers of the senescent cell phenotype. Although it is clear that downregulation of cathepsin X induces cellular senescence, the underlying mechanism remains unknown. The authors proposed that transcription-based events dependent on the expression of cathepsin X are necessary for the reversal toward cellular senescence. In a recent study on human prostate carcinoma cells PC3 they suggested that the mechanism may involve regulation of the insulin growth factor system that is a known activator of tumor cell proliferation [85].

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4.5. Induction of the epithelial–mesenchymal transition

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Cathepsin X may contribute to tumor progression by inducing the epithelial–mesenchymal transition (EMT) [11]. EMT is a biological process allowing polarized epithelial cells to undergo multiple biochemical changes that enable them to assume a spindle-shape mesenchymal cell phenotype. This includes enhanced migratory capacity, invasiveness and increased resistance to apoptosis [86]. In cancer, EMT has been recognized as an important step in the progression of malignant disease toward tumor metastasis. In hepatocellular carcinoma cells QGY-7703, the overexpression of cathepsin X was associated with upregulated mesenchymal markers, such as N-cadherin and fibronectin, and downregulated epithelial markers E-cadherin, ␣-catenin and ␤-catenin [11]. Moreover, cathepsin X upregulated matrix metalloproteases MMP2, MMP3 and MMP9, typical mesenchymal factors, capable of remodeling ECM. Transfection and overexpression of cathepsin X, causing EMT, significantly promotes cell migration, invasion and initiation of metastasis. No role for cathepsin B or other cysteine cathepsins in EMT has so far been reported.

5. Conclusions and future perspectives It is widely accepted that overexpression, increased enzymatic activity and mis-localization of cysteine cathepsins are associated with pathological processes leading to tumor progression. Until recently, the molecular mechanism linking their excessive proteolytic activity with aggressive tumor phenotype was believed to be the degradation of ECM. In particular, cathepsin B and aspartic peptidase cathepsin D have been proved to play an important role in these processes. However, there is increasing evidence that cathepsins are involved in a series of other tumor promoting functions, an important one being the regulation of cell signaling. In particular, cathepsin X, as a carboxypeptidase, has functions other than ECM remodeling. Its interaction with integrin receptors in proteolytically dependent or independent manners, strongly associates its tumor cell function with signal transduction, resulting in changes in cell adhesion, migration, cytoskeleton remodeling and cell proliferation. The proteolytic processing of other targets, such as profilin 1, further emphasizes its tumor promoter function. Bypassing cellular senescence and inducing epithelial–mesenchymal transitions indicate the involvement of as yet unknown molecular mechanisms. Correlation of higher cathepsin X levels with the survival of cancer patients and with their response to chemotherapy form the basis of its possible application as a prognostic and predictive marker for improving the efficacy of existing therapies. On the other hand, cathepsin X is a validated target for the development of new anticancer drugs. Effective control of cathepsin X activity and consequently its tumor promoter function could be achieved by the use of specific protease inhibitors. In contrast to other cysteine cathepsins the list of cathepsin X inhibitors is rather short. Cystatins, endogenous inhibitors of cysteine proteases, are nonselective and bind cathepsin X much more weakly than other cysteine cathepsins. Of the small molecule synthetic inhibitors, epoxysuccinyl ones have been reported to inhibit cathepsin X [33,34,87], although they lack the required selectivity. Neutralizing 2F12 mAb was developed as a highly selective cathepsin X inhibitor [20,34] but it acts predominantly on the extracellular fraction of active cathepsin X. Future work needs therefore to be focused on identifying small, selective and reversible inhibitors, capable of reducing the carboxypeptidase activity of cathepsin X in particular processes related to tumor progression. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements The authors sincerely acknowledge Prof. Roger Pain for the critical review of the manuscript. This project was supported by Research Agency of the Republic of Slovenia (grants P4-0127 and Q5 J4-4123 to JK). References [1] Rawlings ND, Barrett AJ, Bateman A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 2012;40:D343–50. [2] Turk B. Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 2006;5:785–99. [3] Puente XS, Sanchez LM, Overall CM, Lopez-Otin C. Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 2003;4:544–58. [4] Kos J, Lah TT. Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer. Oncol Rep 1998;5:1349–61 [Review]. [5] Schmitt M, Jaenicke F, Graeff H. Protease, matrix degradation and tumour-cell spread. Fybrinolysis 1992;6:1–17. [6] Koblinski JE, Ahram M, Sloane BF. Unraveling the role of proteases in cancer. Clin Chim Acta 2000;291:113–35.

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