diagnosis, follow-up and the anticancer therapy has also been proposed. Contents. 1. ..... in lung cancer patients (54) and in ascites fluid of ovarian carcinoma patients ... more, this offers a potential clinical application of Cat B measurements in ...
ONCOLOGY REPORTS 5: 1349-1361, 1998
Cysteine proteinases and their endogenous inhibitors:
Target proteins for prognosis, diagnosis and
therapy in cancer (Review)
JANKO KOS 1,2 and TAMARA T. LAH 3 1Jozef Stefan Institute, Department of Biochemistry and Molecular Biology, Ljubljana; 2KRKA d.d., R&D Division,
Department of Biochemical Research and Drug Design, Novo mesto; 3National Institute of Biology,
Laboratory for Molecular Biology and Biochemistry, Ljubljana, Slovenia
Received July 10, 1998; Accepted August 31,1998
Abstract. Lysosomal cysteine proteinases, the cathepsins (Cats) belong to the papain family of proteinases, sharing a similar protein structure and mechanism of action. Subtle structural differences between these enzymes give rise to important variations in substrate specificity and specificity of inhibition by their endogenous inhibitors, the cystatins, stefins and kininogens under physiological and pathological conditions. Alterations in their expression, processing and localization have been observed at various levels in malignant human tumor tissue compared to nom1al and benign tissue counterparts. We have proposed that an imbalance between cathepsins and cystatins , associated with metastatic tumor cell phenotype, may facilitate tumor cell invasion and metastasis. The results of clinical investigations on cysteine cathepsins and their endogenous inhibitors in human breast, lung, brain and head and neck tumors, as well as in body fluids of ovarian, uterine, melanoma and colorectal carcinoma bearing patients, have shown that these molecules are highly predictive for the length of survival and may be used for assessment of risk of relapse and death for cancer patients. Their application for ( diagnosis, follow-up and the anticancer therapy has also been proposed.
Contents 1. Introduction 2. Cysteine proteinases 3. Alterations in expression, trafficking and localization of cysteine pl'Oteinases in tumor cells
Correspondence to: Dr Janko Kos, Jozef Stefan Institute , Department of Biochemistry and Molecular Biology, Jamova 39, 1000 Ljubljana, Slovenia Key words: cathepsins, stefins, cystatins, cancer, prognosis, diagnosis, serum
4. Endogenous inhibitors of cysteine proteinases: structure, function and physiological role in normal and malignant cells and tissues 5. Clinical relevance of cysteine proteinases and their inhibitors as indicators for prognosis 6. Conclusions
1. Introduction The ability of tumor cells to invade extracellular balTiers and to metastasize to distant sites is associated with the activity of proteinases. Under nOimal physiological conditions proteinases participate in a variety of processes at tissue, cellular and systemic level. They mediate intracellular and extracellular protein turnover, regulating the lifetime of proteins and other molecules critical for normal cell function. Beside protein turnover, proteinases are involved in specific processing steps for smaller peptides, pro-enzymes and pro-hormones (1). Dissolution and remodeling of connective tissue and basement membrane in the processes of tumor invasion presumably require the concerted action of various intra and/ or extracellular proteinases (2). These include matrix metallo pl'Oteinases (MMP) , serine proteinases, such as urokinase and tissue types of plasminogen activator (uPA, tPA) and plasmin , aspartic proteinase cathepsin D and cysteine proteinases cathepsins B, Hand L (3). All these proteinases are thought to participate in degrading of extracellular matrix proteins in a cascade-like manner (2). Matrix metalloproteinases (MMPs) including collagenases, gelatinases A and B (MMP-2 and 9) and stromelysin (MMP-3) are capable of extensive degradation of collagen triple helixes, as well as of other protein components of extracellular matrix and basal membrane, such as fibrin, fibronectin, laminin and proteo glycans. It has been proposed (2) that latent form s of metallo proteinases can be activated by plasmin, converted from plasminogen by the proteolytic action of uP A and tP A. Plasmin activation presumably occurs on the tumor cell surface, being localized to plasmin and urokinase receptors. Precursor form of uPA can be activated by proteolytic action of numerous enzymes, including cysteine proteinases (4). The activation of cysteine proteinases requires acidic pH and may occur
KOS and LAH : CATHEPSINS AND CYSTATINS IN CANCER
Figure 1 Intracellular localization of Cat B in normal ki dney (A) and in breast tumor tissue (B) by illlnlull ogo id labeling, using l EI MAb (KR KA, d.d.) . Arrows indicate Iysosomes (L): M, mitochondri a; N, nucleus. The figure s were provided by Dr Rok Ro mih , In stitute of Cell Biology , Medical Faculty of the University of LjUblj ana. Slove ni a and Dr Endre Kalman, Medi ca l School or the Uni versity of Pees , HLlngary.
autocatalytically or by other lysosomal cathepsins, such as cathepsin D, in ly sosomal vesicles, locali zed close to the plasma membrane (5). An increasin g number of s tudi es are providing evidence for clini ca l relevance of proteinases in cancer. Protei nases are therefo re attractive targets for drug devel op me nt for thera pe utic appli c ati o n. Advan ces in understanding the stru cture and catal ytic mechanisms of num erous proteinases ope n up the pos sibi lity of designin g new inhibitors. A significant contribution in this direction has been made recen tl y with some sy nthetic inhibitors of metalloproteinases, such as batima stat and marimastat (6 ,7). The studies in vo lving the i nhi bi tors of cysteine proteinases are less ex te nsi ve and ha ve been performed on preclinical level on ly , but this type of inhibitor also has the potential to suppress tumor growth and invasion (8,9). The use of novel inhibitors in clinical practice is dependent not just on their specificity and effecti veness but mainly on the knowledge of their precise role in mechan isms of the proteoly tic system in the development of malignant disease (10). On the other hand, the ability of proteinases to enhance tumor growth, in vas ion and meta stas is designates these molecules as prognostic indicators in ca ncer. Urokinase type of plasmin ogen activator (uPA) was the first proteinase shown to be a prognostic marker in human cancer (3) . Its increased prote oly tic activity, protein co ntent and immunos taining corre late with shorter disease-free interval and with overall survival in many cancer types (2 ,3). The inhibitors of uPA, PAI-1 and P AI-2, and the uPA receptor (uPAR) als o show prognostic sign ificance . In contrast to uPA where high tumor level s are associated with poor outcome of the disease, high level s of tissue type of plasminogen activator (tPA) correlate with favora ble prognosis (11). Cathepsin D was the first lysosomal enzy me suggested to predict the ou tco me of malignant di sease (12-14). High levels
have been shown to correlate with poor progn osis in various tu mo rs, althou g h some of these results were conflicting, particularly wh en ca thepsin D wa s de tected immun o his to chemically (15,16). Cysteine proteinases cathepsins B, H and L have also been suggested to correlate with tumor progression (17). An increasing number of studies are now giv ing evidence that this gro up of enzymes and their inhibitors may also provide significant prognostic information in certain types of cancer (18). We review recent findings on the role of cathepsins B, H and L and their endogenous inhibitors stefins and cystatins in m alignant progression , with particular emphasis on current status of these protein s as biological prognos tic markers in human cancers.
2. Cysteine proteinases Six major families of cysteine proteinases, such as the papain family, ca lpains, c\ostripains, streptococaL cysteine proteinases, viral cysteine proteinases and, more rece ntly , the caspases [also named interleukin 2-converting e nzymes (ICE) and apo pains] comprise the superfamily of cysteine proteinases. Cysteine proteinases of the papain fa mily are mostly loca li zed in Iysosomes (Fig. 1), comprising the cysteine ca theps in s Cat B, Cat L, Cat H, Cat S and many recently discovered members Cat K, Cat 0 /2, Cat F, Cat Wand Cat U (10,19). The confo1ll1ation of cysteine cathepsins are similar to papain, although subtle differences in s tructure make these enzymes v ery distin c t with respect to th e ir s ubstrate s pec ificity, inhibition by protein and synthetic inhibitors and specifici ty in their regulation. In ge neral, cath eps in s are synthes iz ed as 30-50 kDa precursor proteins, which are glycosylated and phosphorylated in Golgi apparatus. Subsequent bindin g to mannose-6 phosphate rece p to rs targets these enzymes to endosomal
ONCOLOGY REPORTS 5: 1349-1361, 1998
vesicles. Although further maturation of these vesicles to primary and secondary lysosomes and the trafficking of cathepsins between these vesicular structures is not fully understood, there is evidence that during these processes cathepsins are trimmed to fully active mature formes) by one or more proteolytic cleavages (reviewed in ref. 20) . Cathepsins differ in all these processing pathways and they are differentially glycosylated , proteolytically processed and targeted to different lysosomal vesicles (21-24). Cysteine cathepsins share a common mechanism of nucleophilic attack by the carbonyl carbon of an amide bond. In the mature protein two catalytic residues Cys 25 and His 159 (papain numbering) form a thiolate-imidazolium ion pair essential for the enzyme activity between pH 3.5 and 8.0 (19). Changes in the ionic strength or pH would affect the protonation of the imidazole ring and thus the activity of the enzyme (25). Reducing agents , such as GSSH, cystine and dithiothreitol may protect and/or reactivate the active site cysteine. In vivo, binding of large protein substrates and extra cellular matrix components may stabilize the enzymes , as suggested for cathepsin B (26-28). The optimum activity of cysteine cathepsins is at slightly acidic pH 5.0-6.5, although they can hydrolyze large protein substrates also at neutral pH. The pH-dependent catalytic activity of cathepsins is rather complex and depends not only of the microenvironment and the substrate conformation, but also on the presence and absence of stabilizing factors (29).
3. Alterations in expression, trafficking and localization of cysteine proteinases in tumor cells One of the most important roles proposed for cathepsins in tumor cell invasion is the degradation of extracellular matrix (ECM). This can take place either intracellularly or extra cellularly. Partially degraded ECM components may be endocytosed and digested intracellularly by heterophagosomal activity of tumor cells, presumably in large acidic vacuoles and mediated by Cat D (30) and/or by Cat B (24). Sloane (24) described two types of breast carcinoma cell lines, BT-20 with dispersed vesicular distribution throughout the cyto plasm , which mediated matrix degradation extracellularly, and BT-549 cell line, where Cat B was observed primarily perinuclear and degraded collagen matrix intracellularly (31). This is in agreement with the previous results of Weiss et al (28), who reported two pools of Cat B, the lysosomal and the plasma membrane associated, the latter confined exclusively to the invasive bladder tumor cells. On the other hand, extra cellular ECM degradation observed at focal points of peri plasma membrane (32,23) may be mediated by cell surface associated cathepsins, representing an alternative mechanism of cathepsin-assisted tumor cell invasion.
Plasma membrane associated proteolysis. Trafficking and targeting of lysosomal enzymes is mostly mediated by mannose-6-phosphate receptor (MPR) pathways. Subsequent to phosphorylation of the high- mannose oligosaccharides which are attached to the I ysosomal enzymes to various extent in the Golgi system, the enzymes bind to MPRs and are transported to an acidic compartment, the early endosome. Acidic environment causes the receptor-enzyme complexes
to dissociate and MPRs to recycle to the Golgi apparatus or to the cell surface (33). Two types of MPRs exist: the high molecular weight receptors (MPR 300) presumably target the enzymes to the endosomaillysosomal compartment and the low molecular weight receptors (MPR 46), which mediate their secretion from the cells. An alteration in these receptors was suggested to effect missorting of lysosomal enzymes and this may also occur in tumor cells (34,35). For example, a decrease and lower affinity of cell membrane associated MPRs was suggested to affect pro-cathepsin B secretion (36) . On the other hand , changes in glycosylation pattern of cathepsin may also impair the binding by MPR. In addition to MPR dependent targeting, carbohydrate independent targeting has been proposed for Cat B in normal cells (37) and human colon carcinoma cell line (38). Recently, Koblinski and Sloane (23) reported on a 70 kDa plasma membrane protein which is able to bind pro-Cat B and this pathway may also play an important role in focal dissolution and subsequent invasion into ECM. A specific membrane pro-enzyme receptors have also been identified for Cat Land Cat D and MPR independent trafficking was suggested for these two cathepsins (39-41). Presumably, these receptors would recognize the pro-parts of cathepsins and may also be impaired in some pathological conditions, including tumor progression (42). Plasma membrane receptors may well account for the cell membrane bound Cat B (31). However, translocation of lysosomal vesicles from the perinuclear region to the plasma membrane, possible fusion and subsequent release of active cathepsin at the cell surface, is an alternative explanation for its cell surface associated activity. Indeed, Cat B was demonstrated on the outer surface of breast cancer cells , MCF-IOA neoT, BT-20 and MCF-7 as well as in brain tumor cells, using confocal microscopy. Translocation of Cat B may be induced in transfonned cells by various stimuli, including oncogene transfection, metabolites of arachidonic acid, irradiation , etc. , although the mechanism of translocation is not fully understood. Presumably , tubulin filaments and mature lysosomal, but not endosomal vesicles, are involved (43). Lysosomal Cat B translocation was also observed in lung carcinoma cells (22) and in LCLH 103 cell line penetrating into collagen gel. Pericellular Cat B was found in the extra cellular face and more often it was concentrated in pseudopodia (32), supporting the observations in breast and brain cancer cells and in murine melanoma cell lines (43).
Extracellular proteolytic activity. The mechanism of secretion of lysosomal cathepsins is also not fully understood (44). Cathepsins were secreted in normal and in tumor cells mainly as the precursor fonns, as demonstrated for Cat B (45-49) , Cat L (48,49) and Cat D (41,50,51), while many types of tumor cells may also release mature, active Cat B (21,52,53). However, it is not known whether the precursors are activated at the plasma membrane or extracellularly. It is possible that a pericellular acidic environment is created , similar to that resulting from active proton transport at the focal peri membrane locations, as observed in activated macrophages and osteoclasts during bone resorption. Subsequent to lowering the pH, cathepsins would be activated (42). On the other hand, it has been demonstrated for example for
KOS and LAH : CATHEPSINS AND CYSTATINS TN CAN CER
Cat B, tha t the enzyme can effectively degrade large protein subst rates als o at ph ys iological pH (26 ,27) and the acidic environment may therefore not be needed for efficient proteo lysis. In keeping with the results in tumor cells in vitro, we may assume that catheps in s are also secreted from tumors in vivo and indeed, they have been found in a vari ety of tumor surrounding fluids, such as bronchoalveolar lavage fluid in lung cancer patients (54) and in ascites fluid of ovarian carcino ma pati ents (55). Also, high lev e ls of cysteine cathepsin s were reported in sera of patients with breast (56), ovarian and uteri cervi x (57), liver (58), pancreatic cancer (59) , colorecta l cancer (60) and m e la noma (61,62). In mel ahoma serum levels of Cat B and Cat H were associated with the progression of th e di se ase (61 ). This confirms previous results (63), which showed that Cat B in serum and urine was high in metastasis bearin g patients, but decreased after tumor removal, indicating either release of Cat B from tumors and/or a systemic respo nse to tumor burden. Further more, this offers a potenti al clinical application of Cat B meas urements in body fluids. In vivo, alteration in subcellular localization could not be observed. However, investigating Cat B immunohistochemica l staining and mRNA by in situ hybridi zation in bladder, colon and prostate carcinomas and in glioblastoma, the enzyme was found more often at the invasive edges, where tumor cells were supposed to degrade basement membrane (23,42). In breast carcinom a, this pattern was observed only occasionall y, while Cat B was st ained heavily in macropha ges around necrotic areas, in myoepithelial cells and in endothelial cells of new capillaries (Lah et ai, unpublished data) (Fig. 2). We found also extensive Cat L staining in the capillaries and macrophages and granular Cat H staining in tumor cells and some capillaries. In brain tumors , Cat B association with endothelial cells was reported (64) and rece ntly we showed that Cat B associated with endothelial cells was related Lo prognosis of patients with glioblastoma (Strojnik et ai, un published data).
c . ~
; " .'
4. Endogenous inhibitors of cysteine proteinases: structure, function and physiological role in normal and malignant cells and tissues Cysteine proteinases are ultimately regulated by endogenous cysteine proteinase inhibitors (CPIs), also named cystatins (reviewed in ref. 65). Cystatin superfamily comprises families of closely homologous CPIs, stefins (family I), cystatin s (family II), kininogens (family III) and non-inhibitory proteins (family IV) , such as human histidine-rich glycoprotein and a2HS-gylcoprotein (66). Structurally more distant members of the cystati n superfamily have been reported , such as p21, the produ ct of c-Ha-ras oncogene, which may be involved in earlier steps of tumor progress ion (67). More recently , p4l frag ment from the invariant c hain (Ii) of the major histo compatibility complex (MHC) II , thyroglobulin type I domain and equinatoxin, an inhibitor from sea anemonae, were discovered, all being CPIs with no close structural homology to cystatins (19). Stefins and cystatins share similar structural features, with molecular size of 11 ,000 and 13,000, res pectively,
Figure 2. Immunohi stoche mical staining of Cat B (A), Cat L (B), and Cat H (C) in paraffin sections of invasive ductal breast carcinoma. The figure was provided by Dr Endre Kalman. CAl, Cat B stain ing in infiltratin g and in silL! compartment of ductal breast carci noma usin g 3El MAb. Arrows indicate Cat B in tumor cells (T), myoepithelial cells (My) su rrou nding ill silu tumor and in infiltrated macrophages surrou nding necroti c area (M). (B), Cal L staining in tumor ce ll nests , in endothelial cells of capillaries (Cp) and in mac rophages (M) by N 135 MAb. (C), Extens ive granular staining of Cat H in tumor cell s and faint stain ing in some capillaries by ID I 0 MAb .
similar folding of the single polypeptide chain and lack of carbohydrate moieties. A significant structural difference betwee n cystatins and stefins is that the latter lack a signal peptide which is respons ible for extrace llular targeting of
ONCOLOGY REPORTS 5: 1349-13 6 1, J998
cystatins, while stefills have been reported as intrace llular inhibitors (65). Based on X -ray c rys tal struc ture of chi c ke n cystatin (68), three region s o f the cystatin mo lecule have bee n proposed as being in cl ose co ntact with the e nzyme, the N-te rminal part and two hairp in loops , the latter containing QVVAG a nd PW motifs, res pectively (68 ,69). Cysteine and/or disulfid e brid ges do not co ntribute to the inhibitOl'y fun ction. This general model of inhibition of cy s teine pro tein ases has bee n confirmed by the crys ta l s tru c ture of s tefin B-papain co mple x (70) and NMR struc tures of chi c ken cy statin (71) and s tefin A (72,73) .
Stef ins. Ste fins are s in g le-chain pro te ins with a m olecular wei g ht of about 1 J ,000 and lackjng dis ulphide brid ges . Human stefin A is an acidic protein with pI values between 4 .5-5 .0 whe reas stefin B is neutral ha ving pI va lu es in the ran ge of 5 .9-6.5. The most si g nifi ca nt differen ce be tween stefin s A and B is lo w e r affinit y o f bindin g of s tefin B [ 0 c ys te ine prote in ases, parti c ul ar ly to C a t B, although no s uff ic ient stru ctural eviden ce fo r this differe nce has been dem onstrated so fa r (73). Al so, the ir tis s ue a nd cellul a r dis tribution is strikjngly different, stefin A bein g localized mainl y to epithelial and lympho id tis sue, while s tefin B is eve nly di stributed in d ifferent ti ss ue cells (74-76) . Stefin A is more resistant to proteolyti c degradation by cathepsin D than stefin B (77). On the bas is of its cellul ar distribution stefin A was pro posed to protect e pithelial and lymphoid ti ss ues from invading bacteria whereas stefin B m ay protec t cells from un co ntroll ed action of endogenou s cyste ine proteases (78). A defect in expression of stefins was associated wi th various diseases, such as cancer, period ontal disease, s kjn Or lung inflamm a ti o n (revi ewed in ref. 79). Mutations in the gene encodin g ste fin B have been found in patients with progress ive myoclonous ep ile psy (80). Cystatins. Famil y II cystatin s are s li g htly larger than the stefin s with a m o lecular we ig ht of abo ut 13,000. They are s ingle c ha in proteins , s ta bili zed by tw o di sulfide brid ges . Human cys ta tin C and its avian analog ue chicken cystatin are the mos t investi gated me mbers of the fa mily II. Cys tatin C is a bun da nt in vari o us ti ssues and bod y fluid s . The highes t levels ha ve bee n de termined in cerebrospina l fluid, semin al plas ma and sy no vial fluid . It is the most po te nt inhibitor of cystein e pro teas es with a pp are nt inhibition con s tants in pi comolar ra nge (8 1). Ch a nges in le ve ls o f c ystatin C in ex tracellul ar fluid s ha ve been shown to be po tentially c lini call y importa nt. Its le ve l in blood pl as ma has bee n sho wn to be a good indicator of g lomerul ar filtra tion rate (GFR), conside rably better than th e widely u se d c reatinin e (8 2,8 3) . D ec rea sed lev e ls o f cystatin C in cerebrospinal t1 uid were sho wn to be associated with he red itary cystatin C amyloid an giopathy (HCCAA) , a domin antl y inherited di sease charac terized by de position of cystatin C variants as amyloid fibrils in blood vessels, resulting in death from cere bral hemorrhage (84). Cy statin C has been suggested to playa role in several other di seases associated with altera ti o ns of the pro teo lytic sy s te m , s Ul:h as ca ncer (79,85 ), inflammatory lun g di seases (86), periodontal d isease (87), multipl e s cleros is (88) , re nal failure (89) , a utoimune diseases (90) and HIV infection (9 1).
Kin inogens. The kinin ogen s are multifunctional pro teins with muc h higher m olec ular we ig hts (50,000-115 ,000) than the pro teins of the fir st two families. They are well known as prec ursors of the vasoac ti ve peptide kinin . In addition , they are involved in the blood coagulati o n cascade, acute phase response, inflammati on and as inhibitors of cyste ine proteases (92 ,93) . Three ty pes of kininogen have been ide ntified : high m o lec ular weig ht kininogen (HK), low m o lec ul a r wei g ht kininogen (LK) and T-kininoge n (TK), the latter being found only in rat. Single chain mature molecul es o f kininoge n are converted to two chain forms by limited pro teolys is by serine protease kallikrein , with release of kinin. Hea vy a nd li g ht c hain s remain connected by a di s ulfi de bond (94). The heavy c ha in s are l:om posed of three cystatin-like d o mains. T wo of the m are a bl e to inhibit cys te ine pro te inases , includin g cathepsins B, H a nd L (95). a2 -macrog lobulin. C ys tei ne proteases are inhibited also by a2-macro globulin (a2M) , the maj or ex tracellul a r sl:a vanger of e ndo-peptidases of all four classes . After initi al proteolyti c cleavage ac ti ve e nzymes are e ntra pped by a2M , bo und to the macrophage receptors and the complexes are cleared fr o m the cir c ul a tion (9 6 ). Th e rate of c leav age of a2 M is pres uma bly related to the e ndopeptid ase activity a nd it is likely that Cat L, whi ch is the mos t powerful cystein e endo pe ptid ase (97), is cleared fa s te r fr om the circu la tio n than C at B, whi c h posses ses end o pe ptidas e and exo peptidase (dipe ptidyl carboxy peptidase) ac tivity , o r cathe psin H , whi c h is mainl y a n e xo(amin o)p e ptidase (20,98). Our recen t studi es support this hypo thesis, revealing increased levels of cathe psi ns Band H in blood o f ca ncer patients a nd their relatio n to prog nos is whereas no such observati on has been fo und fo r cathe psin L (60,61) . Inhibitors of CP in malignant prog ression. S tefin A was the first cy statin re ported to be assoc iated w ith malignant tum ors. Loss of stefin A immun oreactivity in de ndritic reti c ulum cell s in fol ic ul ar lympho mas (99) and in Reed-Sternberg cells in Hodg kin's disease (100) corre lated with tumor ma lignan cy. Decreased immun oreactivity was a lso reported in mali gnant human tum ors of squam ou s e pithelial ce ll origin (74 ,76). Furthermore, during progression of murine skin papillo mas to carcino mas, stefin A mRNA levels decreased (1 01) . Kartasova et al (1 02) observ ed a lte ration of stefin A ge ne in human epid erm al kera tinocy tes in the earl y step of induced carcino genesis. The hypothesis of inverse correlation of stefin A expressio n with metastatic potenti al of breast tum ors was first su gges ted by Lah et al ( 17), who fo und lo wer mRNA levels of stefin A in some, but not in all tum or sa mpl es. Recentl y , the same authors reported the association of lowe r level s of stefin A in tumo r compared w ith normal m a tc hed ti ssue of the sam e breast, with early relapse of breas t cancer [( I 03), Levicar et ai, unpubli shed da ta]. Recentl y, immunohi stoc he mical studi es revealed that in breast tum ors stefin A was not only assoc iated with basal cell s of gla ndular epithe lium ( 104), but was also present in myoepithe lial cells of non- neo plastic du cts, granulo cytes, hi sliocytes and dendriti c re ti culum cell s (Grazio el ai, unpubli s hed data) . However, these data are no t in agreem e nt with an immun o his tochemi ca l stud y of pros tate ca rcin o ma,
KOS and LAH: CATHEPSINS AND CYSTATINS IN CANCER
where stefin A was present in basal epithelial cells of the normal prostate, while in hyperplastic prostate the expression of s tefin A was decreased (100). Kuopio el at (104) also speculated on the role of stefin A in apoptosis as apoptotic bodies consistently stained for inhibitor, which also correlated with p53 activation. Stefin A was purified from his tiosarcoma in a molecular form with lower inhibitory potential compared to liver stefin A, indicating possible mutation in the protein, which may affect its activity (105). In brain tumors, stefin A was present in very low concentrations , when detected immunohistochemically (Strojnik ef at, unpublished data) . Protein le vels of stefins A and B were evaluated also in cytosols of head and neck (106) and I ung tumors (107). In head and neck carcinoma, the media n co ncentrations of stefins A and B were not significantly higher than those in adjacent normal tissues and did not correlate with clinical indicators of tumor progression . However, the levels of both stefins were significantly higher in loc ally advanced than in early tumors (108). Similarly to breas t carcinoma, medi an protein levels of both stefins A and B were significantly higher in lung tumors than in normal lung ti ssue. Cystatin C, a physiologically important inhibitor of extra cellular cysteine proteinases, was sec reted in signi ficant amounts from lung (48) and colon carcinoma cell lines (47). Stefins can also be secreted into body fluids, such as urine and serum. Serum levels of stefin A increased in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC) a nd correlated with tumor size and with the number of neoplastic les ions in HCC patients (58). Serum level s of stefin A were not significantly changed in melanoma patients, while cystatin C was higher than in nonnal sera (61). Cystatin C level s correlated with the stage of di sease, being the highest in metasta tic melanoma patients . In sera of patients with colorectal cancer the level s of stefin A and cystatin C were moderately increased , whereas stefin B levels remained un changed when compared to control s (Kos et at, unpublished data). In the same study stefin A and cys tatin C levels were ind ependent of Dukes' stage, whereas stefin B level s correlated significantly with Dukes ' stage, being the highest in stage D. Siewinski et al (109) found tha t the ratio be tween active an d complexed forms of inhibitors of cys teine proteinases in serum conelated with tumor aggressiveness in head and neck ca ncer. For these patients higher level s of free inhibitors were associated with their better clinical status.
Table 1. Prognostic relevance of cysteine proteases and inhibitors in various cancers. Author/Ref.
Breast Lah ef at (17) Thomssen et al (112) Budihna et at (114) Lah et at (l03)
No Method of pats.
45 167 62 60
Thomssen etat (113) 103 Foeke ns ef at ( I 15) 1500 Kuopio ef at (l 04) 384 Grazio et ata 250 Lung Knoch et a t (117) Ebert et at (l16) Inoue et at (119) Sukoh et at (118) Werle et at(121 )
Werle et at (122) Ebert et at (107 ) Werle et ata Head and neck Budihna el at (108) Smid et at (123) Colon and re ctum Campo el at (141) Kos et at (60) Kos et at" Mel a noma Kos etat(61) Brain Strojnik et at"
EA ELISA ELISA ELISA ,
Cat Lt , Cat Bt Cat Bt Stefins AL, B1-, Cat BT
IHC ELISA ELISA IHC IHC
Cat LT Cat Bt, Cat Lt Ste fin AT Stefin AT
69 EA 65 EA 142 IHC 108 IHC 77 ELISA 91 EA 77 ELISA 127 IHC
45 ELISA 25 ELISA
Cat BT Cat BT CatBT Cat BT Cat LT Cat BT Stefin B1 CatBT
Stefins A1-, B1-, Cat BT Stefins A1-, B1
101 325 349
IHC ELISA ELISA
CatBT CatBT Stefin BT, cys tatin Ci
Cat BT, Cat HT
EA, enzyme activity; ELISA, enzyme-linked immunosorbent assay; IHC , immunohistochemical analysis; 1', correlation of high levels with poor prognosis; 1, correlation of low levels with poor prognosis; "unpublished data.
5. Clinical relevance of cysteine proteinases and their inhibitors as indicators for prognosis The development of metastases is the major cause of death in patients with solid tumors . In many carcinomas, the primary treatment, i.e. the removal of the tumor, is followed by adjuvant systemic therapy. Application of the therapy is based on the assessment of risk for relapse or death, dependent on the presence of systemic micrometastasis. Prognostic factors allow distinction between high and low-risk patients. Selection of patients for toxic adjuvant therapy may improve its effect on hi gh-risk patients and avoid unnecessary treatment of low risk patients (110).
Several esta blished clinical and histopathological factors are used for predic ting prognosis, and include tumor size, lymph node involvement, tumor cell morphology, etc. which di scriminate between low and high-risk patient populations . However, in low-risk populations, a substantial number of patien ts still e xperi e nce relapse wi thin 10 years after the operation (II I). Therefore, for these patients new biological markers are needed, which are related to the malignancy and
ONCOLOGY REPORTS 5: jJ49-1361 , 1998
Table II. Tumor-as soc iated extracellul ar leve ls of cysteine prote in ases and their inhibito rs. Cancer
Cats B, H, L
Colon and rectum
Cats B, H , L , CPI stefins A , B, cystatin C
Bronch o- Cat B , CPI alveolar Fluid Serum Cat B
Ascitic fluid Se rum S erum
CPI, ste fin s A , B
CatL Cat B
Pan c reas
Cat B , Cat L
Cat B, Cat L, stefin A
Head and neck
Cat B, CPI
Cat B, Cat L, Cat H. stefin A, cystatin C
M e lanom a Serum
to the metastatic potential of the primary tumors. Among these biological fac tors are cysteine proteinases and their inhibitors (Table I). Various techniques have been applied to determine the expression of cysteine proteinases and their inhibi to rs in tumors . The se in c lud e measurements of e n zy matic or inhibitory activities us in g synthetic a nd nativ e substrates, mRNA qu a ntitation and application of va rious poly clon al and mon oc lonal antibodies in ELlSAs, immunoblots and immun ohi s tochemical anal ys is. In mos t of the studies tiss ue cy tosols, prepared from primary tumors have bee n used as clinical samples. M eas urement of the same parameters by different as says a nd procedures led to sig nificantly varied results, indicating the need for qu a lity control and s ta nda rdizin g protocols before intro du c ing these new prognostic indicators into c linical practice. As described above, cathepsins, stefins and cystatins can also be secreted and hav e bee n found in biolog ica l fluids surroundin g tumors and in serum and urine of tumor patients (Table II). Their quantitation in extracellular t1uids is much more sim pl e th an in tissues , and wo uld provide a m o re co nvenient route to prognos is, mo nitoring the course of the disease and checking theeffectiveness of the adjuvant therapy. The level of proteinases a nd inhibitors in extracellular t1uids reflects not only the ir loc al expression in tum ors but also the systemic respo nse to the disease. The concentrations of cathepsins and stefins in blood and other t1uids are significantly lower than in tumors and their quantification requires sensitive
assays. The latter is probably the reason why the study of the association of serum or plasma levels of cysteine prote inases a nd their inhibitors with malignancy and prognosis has been limited. Breast cancer. Cat D was the first of the lysoso m al enzymes
proved to be of prognostic sig nifi cance for survival of breast cancer patients, reviewed by Rochefort et af ( 14). Lah et af (17) found that Cat B and Cat L act ivities were much more elevated in breast tumors compared to Cat D and s uggested that Cat L might be of prognostic s ignific anc e. Thi s was confirmed la ter by Thomssen et al (112) who found that increased Cat L protein level in tumor ti ssue cy tosols was a stro ng and independent prognostic factor with an imp ac t co mparable to that o f axillary lymph node s tatu s and grading. The same investigators (113) recentl y showed that a low lev e l of Cat L in combination with a lo w leve l of plasmino ge n ac tivator inhibitor 1 (PAI-l ) is particularly useful in ide ntifying lymph nod e negativ e patients with an extremely good prognosis. In co ntras t to the previous results, Budihn a et af (114) reported th at a group of breast ca ncer patients with low Cat B levels ex perie nced poor survi val. In a rece nt study of m atched pairs of breast tum o rs and co ntrol breast tissues, increased activity and prote in levels of Cat B proved a better· prognostic indicator value than those of Cat L, while Cat D increase did not (103). In a lymph node negative population, Cat B protein and activity were better prognostic indicator for disease-free surviv al than in the total pati e nt population. In this study a large inc rease in Cat B and Cat L enzymatic activities was measured relative to thei r protein concentrations, indi ca ting highly e nhanc ed activation of cath eps in s in tumors. The latter may be due either to the e nha nced activation of their precursors and/or to the down regulation of the endoge nous inhibitors. Indeed , a signific ant decrease of stefin A level in some, but not all tumors was observed , as sugges ted previously (17). In the res t of the tumors a mod e rate increase of s te fi n A was observed. Howeve r, patients with less increased stefin A experienced poorer rates of survival (Levicar et ai, unpublished data). The data stro ngly suggest that pati ents with increased levels of cyste in e cathepsins in tumors, with no compensating increase in s tefin levels, have a poorer prognosis than patients with higher levels of stefin s. Immunohistochemical analysis of stefin A in breast tumors rev ealed a n opposite relation to survival compared with the studies on activity and protei n level. Kuopio et af (104) fou nd tha t the risk of death was significantly higher in patients wi th positive staining of stefin A in tumors. The increase in a ri sk was also signifi cant in lym ph node negative patients. Further more , co-expression of stefin A and p53 was indicative for hi g h-ri sk pa tie nts. Hi g her staining of s tefin A was also found to be associated wilh poor prognosis in our m ost recent study on 250 breast tumors (Grazio et ai, manuscript in preparation). In the recent study of Foekens et af (115) on 1,500 patients with primary breast carcinoma, bOlh , Cat B and Cat L levels were found to be effective in predictin g the rate of relap se and the length of survival after remova l of the primary tumor. The levels, measured in tumor ti ss ue cy tosols, were positi vely corre lated with the numb er of positive ly mph nodes and
KOS and LAH: CATHEPS INS AND CYSTATINS IN CANCER
negatively with the level of the steroid-hormone receptors. No significant relatio nship was found between Cat B and Cat L levels and age, me nopa usa l status of the patients and size of the primary tumor. The levels of Cat B an d Cat L positively corre lated with eac h o ther and with the relapse-free and over all survival in univariate analysis. In multivari ate regression analysis, corrected for age, menopausal sta tu s, tum or s ize, th e number of invaded lymph nodes a nd steroid -horm one receptor statu s, Cat B and Cat L were found to be s ignificant independent factors for prediction of the rates of re lapse and dea th .
Lung carcinoma. In lung carcinoma, Cat B activity and the rati o between Cat B and inhibitory activ ity were significant for prognosis (116,117). Recently , we have confirmed the imbalance be tween these antagonistic mo lecules and reported the prognostic significance of stefin B in lung cancer patients. However, the expression of cys teine cathepsins and stefin s seems to vary with tum or hi sto logy (107). Using immuno histoc he mica l analysis of non-s mall ce ll lun g tumor ti ssue sec ti ons Sukoh et al (I ( 8) a nd Inoue et al (119) found the association of higher Cat B with shorte r survival , whereas M ori et al (120) have found no correlation between survival and Cat B expression. The latter is co nsistent with the results of the recent study on 77 matched pairs of tumors and nonn al lung parenchyma establishing the association with tumor progression only for Cat L but not for Cat B concentration (121). It has b ee n s ugges te d , th a t amo ng vario us Cat B fractio ns present in lung tumors and nOimal lung tissue, onl y the fraction acti ve at pH 7.5, provides prognostic inform ation in patients with squamous cell carcino ma ( 122). In the most recent stu dy (Werle et ai, unpublis hed data) we found that the positive s taining of Cat B in tumor cells correlated significantly with the survival of patients with squamous ce ll carc inoma. Head and neck carcinoma. In 45 matc hed pairs of tumors and adjacent normal tiss ues of patients with head and neck cancer, the median leve ls of Cat B, Cat L and Cat D were significantly hig her in tum ors than in normal tissues (106). In co ntrast, Cat H was signifi cantly decreased in tumor tissues. The increase in cathepsins B, Land D and the decrease in Cat H a nd stefins A and B co rre lated with a poorer prog nosis ( 108). The levels of stefins A and B were higher in locally adv a nced than in early tumors. However, patie nts with lower tumor levels of stefin A showed s ignifi cantly shorter di sease free and disease s pecific survi val times than the patie nts with rugh levels, similar to those observed for breast cancer patients. In additio n, in head and neck cancer patients, stefin B was also a s ig nifi ca nt progn ostic indicator for di sease-free and disease-specific survival ( 108, 123) . The differences found in breast, lun g and head and neck carcin o ma in the prog nos tic value of stefin s may be due to different cellular origin of stefins (76) and the involvement of these ce lls in tumor progression. Conflicting results between immunochemical analysis by ELISA and immun o hi s to che mical s taining of stefi ns suggest that various types of cells are involved and further stud ies are needed to understand the association of stefin A with mali gnant cell differe ntiatio n in carci no mas.
Brain tumors. Several studies ha ve repOited the correlation of Cat B with progre ss io n and inva sio n of human gl ioma (64,124,125). Sivaparvathi et al (126, j 27) demonstrated that Cat L and Cat H are a lso involved in the process of gl ioma progression. Their res ults suggest that the ex pression of both ca thepsi ns is up-reg ul ated in gl iob lasto ma and anaplastic as trocy toma compared with normal brain tissue and low grade g li omas. Recently, using immunohistochemistry, we found hi g h expression of Cat B protein in tumor cells and in endothe li al cell s, for both significantly hi g her in malignant tumors vs. benign tumors (Strojnik et ai, unpubli shed data). Immun o stai nin g of Cat B correlated with se veral clinical features, such as the duration of symptoms, Karnofsky score, psycho organic symptoms and hi stologica l score. Univariate survival analysis indic ated that hi g h Cat B score in tumor a nd e ndothe li al cells was a sig nifi ca nt predic tor of patients' survival. Interestingl y, in gli oblastoma multiforme, hi gh Cat B score in endothelial ce ll s was the most s ig nificant for prediction of su rvival time. Low express ion of stefi n A was found in brain tumors, the immunostai nin g for stefin A was observed only in a few tumor cell s of benign tumors, but not in malignant brain tumors . Tumors of genital tract. Increased levels of Cat L were found in sera of patients with ovarian carcinoma (128). Also, ovarian tum or samples ex pressed hi gher levels of Cat L mRNA th an those of uterin e tumors, benign ova ri a n tumors or normal ovary sampl es. It has been suggested that serum levels of Cat L, in combination with CA 125 a nd CA72-4, may be more useful in detecting ovarian cancer tha n the methods curre ntl y used in clinical practice. Cat B activity, dete rmined in serum was s uggested as a factor for estimating the ex tent and spread of uterin e cervi cal carcinoma (57,129,130). Activity of this enzyme in creased both in serum and in tumors with the progress ion of the disease and was dependent on the clinical stage o f cervical carcino ma . Additionally, the co ncen trati on of Cat B in seru m was found to correlate with different modes of treatment of patients with uterine cervica l carcinoma ( 131 ).
Melanoma. We measured the serum levels of Cat B, Cat H, Cat L, Cat D , stefin A and cystatin C for a group of 98 patients with malignant melanoma (61,62,132). Cat B was signi ficantly in c reased in sera of pati e nts with metastatic melan o ma compared with patients with no metastasis and with control s (Fi g . 3). Cat H was also s ignifica ntl y increased within the gro ups of metastatic and non -metastatic patients. Additionally, Cat H was increased more within the grou p of patie nts with no response to chemotherapy co mpared with the gro up of the responders (6 1). The potential of this enzyme to predict the effecti veness of the therapy could be of considerable clinical importance for selecting treatm ent for individual patients. The concentration profile of serum cystatin C was sirrlil ar to that of Cat H , being the hi g hest for metastatic mel a no ma pati e nts, whereas Cat D , Cat Land stefin A were not significantly changed among the gro ups of controls, no n me tastatic and me tastatic patients. In univariate and multi variate ana ly sis, both hig h Cat B and Cat H values cOlTelated s ig nifi cantly with s horte r overall su rvival of metastatic melanoma patients and seem to be stro nger factors in predicting
ONCOLOGY REPORTS 5: 1349-1361, 1998
0 Cat B
~ non-metastatic melanoma
• metast. melanoma
~ colorectal cance~ l
Figure 3. Concenlration profile of Cal B, Cat H, stefin A and cyslalin C in sera of melnnoma and colorecral pntienls and in healthy cOl1lrols. determined by ELiSAs. The mean levels, oblained for cOl1lrol sera, have beel1110rmalized to a relative concentralion of 1.0.
the length of survival than some clinical factors, suggested as prognostic indicators for melanoma patients (133,134) such as age, gender, tumor thickness, histological type and lymph node metastasis (61).
Colorectal cancer. Increased enzymatic activity, specific for Cat B and its messenger RNA content have been found in several tumors of colon and rectum (135-139). In some of these studies (136,138-140), tumor specific increase was found to be greater in earlier stage tumors (Dukes' A and B) than in more advanced tumors (Dukes' C and D). Regarding enzymatic activities, specifically determined in individual matched pairs for Cat L, Cat B and matrix metalloprotease-9 (MMP-9), Murnane et al (139) suggested four proteolytic profiles, designated as 'early', 'middle', 'late' and 'high'. Significantly decreased levels of Cat B and Cat L were typical for 'late' profile. These profiles should provide information independent of Dukes' stage and may identify crucial variations in tumor behaviour (139), however, their association with prognosis will have to be investigated. Immunohistochemical studies (8,14 I) demonstrated the opposite results, i.e. increased expression of Cat B was demonstrated in advanced stage colorectal tumors. Additionally, high levels of Cat B were shown to be associated with shorter survival of patients (141). Increased Cat B activity was also shown to correlate with colonizing potential (85), metastatic capability (63) and differentiation (38) of various colon cancer cell lines, indicating additionally the involvement of Cat B in later events of tumor progression. Recently, we determined Cat B levels in 325 preoperative sera of patients with colorectal carcinoma (60). A significant correlation between increased Cat B levels and more advanced Dukes' stages was found. Cat B levels were the highest in Dukes' D patients. In survival analysis patients with high Cat B serum level experienced significantly lower survival probability than patients with low Cat B levels. When the data for serum Cat B were combined with carcinoembryonic antigen (CEA) levels, a significant improvement of prognostic
impact has been obtained, i.e. patients with high levels of both molecules had very poor prognosis. For the same patient population the levels of stefin A and cystatin C were found to be independent of Dukes' stage, whereas stefin B levels correlated with Dukes' stage, being the highest in stage D (Kos et ai, unpublished data). In survival analysis stefin B and cystatin C were found to be significant predictors of survival. Patients with high serum levels of stefin B or cystatin C had significantly shorter survival times than patients with low levels of both inhibitors.
6. Conclusions Numerous studies have provided evidence that cysteine proteinases cathepsins B, Hand L are linked to remodeling and degradation of tumor tissue and surrounding extracellular matrix proteins. The levels of these enzymes and their inhibitors in tumors as well as in some extracellular fluids are associated with tumor progression and with disease-free and/or overall survival and, therefore, may serve as prognostic factors for cancer patients. For some cancers these new bio logical markers have also been suggested to aid diagnosis and to assess the response to the chemotherapy. However, further studies on larger patient popUlations, using standardized chemical and immunochemical methods are needed to confirm clinical relevance of cysteine cathepsins and inhibitors for diagnosis and prognosis in cancer. It is necessary as well to define the subpopulations of patients, who would benefit most from more accurate information provided by these new biological parameters alone or in combination with other established diagnostic and prognostic factors. In addition, further knowledge of the precise biological role of cysteine proteinases in the development of malignant disease, based on basic research and clinical investigations may suggest new therapeutic strategies, including targeting to balance the increased tumor associated cysteine proteinases by their inhibitors.
KOS alld LAH : CATHEPSINS A ND CYSTATINS IN CANCER
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