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Research paper
Dual contrasting roles of cysteine cathepsins in
cancer progression: Apoptosis versus tumour invasion
Olga Vasiljeva*, Boris Turk*
Department of Biochemistry and Molecular and Structural Biology, J. Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia
Received 20 July 2007; accepted 12 October 2007
Available online 18 October 2007
Abstract
Cysteine cathepsins have been known for a long time to play an important role in cancer progression and metastasis. Several studies have
proposed the concept of anti-cathepsin therapy in cancer treatment. On the other hand, cysteine cathepsins have been recently found to play
a role in tumour cell death through mediation of apoptosis. The purpose of this mini-review is therefore to provide an insight into the mech-
anisms by which cysteine cathepsins modulate apoptosis and/or participate in tumour invasion, and to evaluate the impact of these enzymes
on both tumour progression and development of potential strategies for cancer treatment.
Ó2007 Elsevier Masson SAS. All rights reserved.
Keywords: Cysteine cathepsins; Cancer progression; Apoptosis; Tumour invasion; Cancer treatment
1. Introduction
In the last decade, it has become evident that multicellular
organisms are protected against redundant and potentially
harmful cells by a highly sophisticated program, termed apo-
ptosis. Apoptosis is a natural mechanism of cellular self de-
struction that is characterized by specific morphological
features, including cell shrinkage, membrane blebbing, chro-
matin condensation and nuclear fragmentation. Apoptosis is
a key process for the development and maintenance of cellular
homeostasis, and acts as part of a quality-control and repair
mechanism by elimination of unwanted, genetically damaged,
or senescent cells and as such is also critically important for
the development of multicellular organisms. In fact, defects
in apoptotic pathways contribute to a number of human
diseases, ranging from neurodegenerative disorders to malig-
nancy [1,2]. The molecular mechanisms that control and exe-
cute apoptotic cell death in cancer growth and resistance have
been coming into focus and are enabling a new era of drug de-
velopment for cancer treatment [3].
A family of cysteinyl aspartate-specific proteases, called
the caspases (Clan CD; C14 family), have a critical role in ap-
optosis [4]. Other proteases have also been found to participate
in apoptosis, among them being cysteine cathepsins (Clan CA;
C1 papain family) [5]. However, cysteine cathepsins are also
well known to play a tumour-promoting function in cancer
progression for example by actively participating in tumour in-
vasion processes [6]. The purpose of this mini-review is there-
fore to provide an insight into the mechanisms by which
cysteine cathepsins modulate apoptosis and/or participate in
tumour invasion, and to evaluate the impact of these enzymes
both for tumour progression and for potential strategies of can-
cer treatment.
2. Cysteine cathepsins
Eleven human cysteine cathepsins have been identified: ca-
thepsins B, C (J, dipeptidyl peptidase I), F, H, K (O, O2), L,
Abbreviations: BH3, Bcl-2 homology domain 3; Bid, BH3-interacting
death domain; ECM, extracellular matrix; MHC, major histocompatibility
complex; uPA, urokinase plasminogen activator; TNF, tumour necrosis factor;
TRAIL, TNF-related apoptosis inducing ligand.
* Corresponding authors. Tel.: þ386 1 477 3772; fax: þ386 1 477 3984.
E-mail addresses: olga.vasiljeva@ijs.si (O. Vasiljeva), boris.turk@ijs.si
(B. Turk).
0300-9084/$ - see front matter Ó2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2007.10.004
A
vailable online at www.sciencedirect.com
Biochimie 90 (2008) 380e386
www.elsevier.com/locate/biochi
Author's personal copy
O, S, V (L2), W (lymphopain) and X (P, Y, Z) [7]. All have
been biochemically characterized except cathepsins O and
W[8,9]. Cysteine cathepsins share a common fold of the pa-
pain-like structure and are synthesized as inactive zymogens
that are activated by proteolytic removal of the N-terminal
propeptide [10]. Most of them are endopeptidases, with cathep-
sins H and B also being an aminopeptidase and a carboxydipep-
tidase, respectively [11,12]. The only true exopeptidases are
the aminodipeptidase cathepsin C and carboxymonopeptidase
cathepsin X [13]. Cysteine cathepsin activity is regulated by
several mechanisms that comprise regulation of synthesis, zy-
mogen processing, inhibition by endogenous inhibitors (e.g.
cystatins) and pH stability (reviewed in [8]).
For many years cysteine cathepsins were believed to be lo-
calized exclusively in lysosomes, however, it is clear now that
cathepsins including their splice variants are also found in
other cellular and extracellular compartments, including the
nucleus [14,15], extracellular milieu [16,17] and plasma mem-
brane [17,18], where they fulfil important functions. Besides
their main function as a lysosomal protein recycling machine,
the cathepsins have been shown to play roles in a variety of
physiological and pathological processes [9,10]. Cysteine ca-
thepsins are involved in major histocompatibility complex
(MHC) class II-mediated antigen presentation by generation
of the antigenic peptides and by processing the MHC class
II invariant chain, thereby participating in the maturation of
the MHC class II complex. Cathepsins also play a role in
bone remodelling, precursor protein activation (e.g. prohor-
mones), keratinocyte differentiation, etc. In addition, cysteine
cathepsins have also been demonstrated to participate in sev-
eral pathological conditions such as tumour progression and
metastasis, rheumatoid arthritis and osteoarthritis, and athero-
sclerosis (reviewed in [9]).
3. Cysteine cathepsins in apoptosis
Two alternative pathways can trigger apoptosis in mam-
mals: one is mediated by death receptors from the TNF family
on the cell surface (referred to as the ‘extrinsic pathway’) and
the other is mediated by mitochondria (referred to as the ‘in-
trinsic pathway’). The extrinsic pathway is initiated at the
cell surface through cytokine-induced death receptor-mediated
activation of caspase-8 (-10) followed by caspase-3 and -7
activation [19]. The intrinsic pathway is characterized by mi-
tochondrial dysfunction, resulting in release of cytochrome c,
followed by apoptosome formation and subsequent activation
of caspase-9 and, subsequently caspases-3 and -7. The latter
caspases, also called the executioner caspases, are responsible
for the morphological changes during apoptosis, including
membrane blebbing, cell shrinkage, and DNA fragmentation.
Both pathways are connected through Bid, a pro-apoptotic
Bcl-2 family member, which can be cleaved by caspase 8,
generating a truncated Bid fragment (tBid) [20] that induces
mitochondrial outer membrane permeabilization (MOMP)
via the multidomain pro-death molecules Bax or Bak [21].
In turn, Bax/Bak translocation to mitochondria induces the
release of cytochrome cinto the cytosol and subsequent acti-
vation of the executioner caspases.
Recently, cysteine cathepsins have attracted considerable
attention as potential mediators of apoptosis. It is well estab-
lished that massive lysosomal rupture would induce necrotic
autolysis of cells (cellular necrosis), a process mediated by
the lysosomal cathepsins and other ‘‘acidic’’ hydrolases. On
the other hand, more selective release of cysteine cathepsins
as a consequence of limited lysosome damage has been sug-
gested to lead to apoptosis, as also demonstrated in L-leucyl-
L-leucine methyl ester -induced apoptosis in HeLa cells [22].
Whereas lower concentrations of this lysosomotropic agent in-
duced apoptotic cell death, the use of higher concentrations re-
sulted in necrotic cell death with no caspase activation observed.
However, despite numerous reports on their involvement in ap-
optosis, the mechanism(s) are still not clear, except that they
need to be released into the cytosol in order to exert their proa-
poptotic activity. Several models for the release of lysosomal
cathepsins release into the cytosol have been proposed, mostly
based on the mode of lysosomal membrane permeabilization
[23,24]. Most of these stimuli directly target the lysosomal
membrane, either through lysosomal destabilization or
through lysosomal membrane permeabilization. These include
bile salts [25], sphingosine [26], reactive oxygen species [27],
polyclonal antithymocyte globulins [28],L-leucyl-L-leucine
methyl ester [22,29], microtubule stabilizing agents [30],
cold ischemia-warm reperfusion [31], or hexadecylphospho-
cholines [32]. Furthermore, it is now well established that ly-
sosomal permeabilization could occur in response to specific
signalling processes induced by death ligands such as tumour
necrosis factor a(TNF-a)[33] and TNF-related apoptosis in-
ducing ligand (TRAIL) [34,35].
Once released into the cytosol, cysteine cathepsins can in-
duce liberation of cytochrome cfrom the mitochondria, which
was suggested to proceed through the cleavage of pro-apopto-
tic factors, such as Bid [36]. It was shown that almost all cys-
teine cathepsin endopeptidases are capable of cleaving Bid in
the flexible loop to its truncated form [22]. Furthermore, ex-
perimental induction of early lysosomal destabilization by
treating cells with the lysosomotropic agent L-leucyl-L-leucine
methyl ester induced cleavage of Bid and cytochrome crelease
from mitochondria [22]. However, alternative pathways have
been proposed [37]. There is also evidence for a role of ca-
thepsins in the execution of cell death independent of caspase
activation, which results in an apoptosis-like morphology
[38,39].
4. Cysteine cathepsins in cancer
Proteases are known to participate in different stages of
cancer progression. The first reports linking cathepsins to can-
cer originate from the 1930s [40] from studies on tumour
transplantation in rats. The second wave of interest for cathep-
sins in cancer occurred in the late 1960s [41,42], when raised
activity of cathepsins was detected in human tumour samples,
and has continued to the present. However, in spite of such
a relatively long history, the mechanisms and pathways for
381O. Vasiljeva, B. Turk / Biochimie 90 (2008) 380e386
Author's personal copy
the involvement of cysteine cathepsins in cancer are still under
investigation. Increased activity and/or expression of cysteine
cathepsins, mainly cathepsin B, has been detected in many hu-
man tumours, including breast [43e45], lung [46,47], brain
[48e50], gastrointestinal [51e53], prostate [54,55] cancer
and melanoma [56,57], especially in aggressive cancer cells
(for a review see [58]). Moreover, increased expression of cys-
teine cathepsins has been shown to have diagnostic and prog-
nostic value for patients with a variety of malignancies [59].
During malignant progression, peripheral redistribution of
lysosomes with subsequent translocation of cysteine cathep-
sins to the cell surface and/or their secretion into the extracel-
lular milieu appear to be general phenomena in tumour cells
[60e64]. Several mechanism for extracellular translocation
of cathepsins were proposed, among them acidification of
the tumour microenvironment, characteristic of many tumours,
was suggested to induce upregulation of extracellular cysteine
cathepsins, mainly cathepsin B [65,66]. Many studies support
the important role of cysteine cathepsins in the remodelling of
extracellular matrix (ECM) in the tumour microenvironment,
thus promoting tumour invasion and metastasis [67e69].
The proteolytic activity of pericellular cysteine cathepsins ap-
pears to be required for (i) direct proteolysis of ECM compo-
nents such as laminin, fibronectin, type IV collagen, (ii)
initiation of the proteolytic cascade by activating other prote-
ases such as pro-uPA (urokinase plasminogen activator) [70]
and (iii) inactivation of cell-adhesion proteins such as E-cad-
herin (reviewed in [6,71]). However, despite the major func-
tion of extracellularly translocated cysteine cathepsins in
ECM remodelling, the intracellular-vesicular proteolysis was
shown to be also important for digestion of ECM proteins
[72e74].
Cysteine cathepsins participate in hallmark tumour pro-
cesses such as angiogenesis, cell proliferation, apoptosis and
invasion [75]. Furthermore, the use of transgenic mouse cancer
models, in combination with deletion of specific cathepsin
genes, provided the possibility of dissecting their individual
roles in cancer development. This approach was used for pan-
creatic islet tumours (RIP1-Tag2) and for mammary cancer
(MMTV-PyMT) transgenic mouse models [63,71]. Both stud-
ies proved the important role of cysteine cathepsins for tumour
progression and invasion, and two important lessons can be
learned. First, individual cysteine cathepsin genes make dis-
tinctive contributions to tumourigenesis. It was revealed by
the investigation of four cysteine cathepsins null mutations
in the same RIP1-Tag2 mouse model that each enzyme has
an individual, important function in key processes such as tu-
mour cell proliferation, apoptosis, angiogenesis and tumour in-
vasion [71]. For instance, cathepsins B and L knockouts
exhibit reduce cell proliferation and tumour growth, cathepsins
B and S impaired tumour angiogenesis, and deletion of cathep-
sins B, L and S increases tumour cell death, while genetic ab-
lation of cathepsin C had no effect on any of those processes.
The second important message is that depletion of one prote-
ase might induce compensatory upregulation of other family
members. Vasiljeva et al. [63] have shown that genetic abla-
tion of cathepsin B in primary PyMT tumour cells induces
membrane translocation of another carboxypeptidase, cathep-
sin X, that restores the invasive capability of tumour cells and
annihilates the effect of the principal protease deletion on the
level of lung metastasis.
Taken together, these results support the potential therapeu-
tic value of cysteine cathepsins as anti-cancer drug targets and
suggest the use of selective inhibitors of cathepsins for tumour
treatment.
5. Cysteine cathepsins in cancer cell death
Apoptosis is a major defence mechanism against malignant
transformation. Defects in the processes controlling apoptosis
can extend the life span of a cell, thereby contributing to neo-
plastic cell expansion independent of cell division. Moreover,
failures in normal apoptotic pathways contribute to carcino-
genesis by creating a permissive environment for genetic insta-
bility and accumulation of gene mutations. Thus, elucidation
of the new cell death pathways will provide new insights
into tumour biology, revealing novel strategies for combating
cancer [3,76].
There is considerable evidence that cysteine cathepsins
play an important role in executing the apoptotic program in
several tumour cell lines induced by death ligands such as tu-
mour necrosis factor a(TNF-a)[38,77] or TNF-related apo-
ptosis inducing ligand (TRAIL) [34]. Cysteine cathepsins
have been shown to participate in TNF-ainduced apoptosis
of human cervical carcinoma ME-180, murine fibrosarcoma
WEHI-S [38], ovarian cancer OV-90, prostatic cancer PC-3,
hepatoma SMMC-7721 [77] cells. In the last few years, an ap-
optosis inducing agent TRAIL that was shown to specifically
target cancer cells without damaging normal cells and tissues
has received special interest in cancer therapy. The presence
of a cathepsin-mediated proteolytic event in the apoptotic
pathway triggered by TRAIL was recently discovered by sev-
eral groups. Initially the inhibition of TRAIL induced apopto-
sis was observed upon treatment of cells with cathepsin
inhibitors in oral squamous carcinoma cells [78]. Subse-
quently, the pathway for apoptosis induction through Bid ac-
tivation was confirmed for several other tumour cell lines
[34,35,79].
The lysosomal pathways of apoptosis are suggested to be
more complex, containing additional components such as
anti-apoptotic enzyme sphingosine kinase 1 that was shown
to be inactivated by cytosol released cathepsin B [80]. In ad-
dition, the human homologue of SETA binding protein 1
[81], bikunin and TSRC1 [77] were shown to interact with ca-
thepsin B in vitro and in vivo and to participate in the lyso-
somal enzymes-triggered pathway of TNF-a- induced
apoptosis.
Despite the well characterized mechanism for the pro-apo-
ptotic function of cysteine cathepsins through cleavage of Bid
and activation of the mitochondrial pathway of caspase-depen-
dent apoptosis, there have been reports suggesting dominant
execution functions of cysteine cathepsins in apoptosis, even
in the presence of pan-caspase inhibitors [38]. However,
some contradictory findings have been published, confirming
382 O. Vasiljeva, B. Turk / Biochimie 90 (2008) 380e386
Author's personal copy
the anti-apoptotic function of cysteine cathepsins, mostly con-
centrating on cathepsin L [71,82e84].
Thus, summarizing the results derived from a variety of re-
ports, it has become evident that cysteine cathepsins are in-
volved in different pathways to apoptosis in a manner
dependent on cell type and cell stress involved. This conclu-
sion is supported by results obtained recently by analysis of
apoptosis in cathepsin B deficient mouse cells. Whereas prote-
ase depleted hepatocytes were resistant to TNF-a- induced ap-
optosis both in vitro and in vivo [33,85], analysis of mammary
tumour cells from cathepsin B knock-out mice did not reveal
any differences either in spontaneous apoptosis in vivo or in
TNF-ainduced apoptosis in vitro (Vasiljeva et al., submitted).
Further, a significant increase in spontaneous apoptosis was
detected in cathepsin B depleted pancreatic tumour cells in
vivo [71]. In all cases, results were compared with those
from the respective cathepsin B wild-type cells. Nevertheless,
induction of lysosomal permeabilization would always result
in release of cathepsins into the cytoplasm where they can trig-
ger apoptotic cell death via various pathways [24] and thus
may prove effective in cancer treatment.
The use of chloroquine, a lysosomotropic antimalaria drug,
might serve as a successful example of such a pharmacological
approach as it has been shown to inhibit cell growth and in-
duce cell death in lung cancer cells [86], Myc-induced model
of lymphoma [87] and was used in clinical trials as adjuvant
therapy for patients with glioblastoma multiforme [88]. An-
other example is the use of lysosomotropic agent L-leucyl-L-
leucine ester, which was shown to be extremely effective in
killing various immune and myeloid tumour cells of bone-
marrow origin and to efficiently protect against graft-vs.-host
disease in a mouse model of bone marrow transplantation
Cytochrome c
TNF-α
TRAIL
Lysosome
Cathepsins
Caspase 8
APOPTOSIS
Mitochondria
Caspase 9
Caspase 3, 7
Bid
Bax/Bak
Caveolae
Collagen
TUMOR INVASION
Intracellular stimuli
ECM degradation
E-cadherin
MMP
Plasmin
uPA
Fig. 1. The multiple roles of cysteine cathepsins in tumour invasion and apoptosis. Depending on their localization, cysteine cathepsins can be involved in two
apparently opposing mechanisms of cancer progression: tumour invasion and apoptosis. Cysteine cathepsins that are secreted or associated with the plasma mem-
brane (e.g. caveolae) participate in tumour invasion through proteolytic cascade activation, ECM degradation and inactivation of cell-adhesion proteins (e.g. E-
cadherin). In addition, intracellular proteolysis by cysteine cathepsins contributes to degradation of endocytosed collagen in lysosomes. On the other hand, cytosol
translocated cysteine cathepsins trigger apoptosis via Bid cleavage followed by cytochrome crelease from mitochondria. This cascade activates downstream ex-
ecutioner caspases resulting in tumour cell death.
383O. Vasiljeva, B. Turk / Biochimie 90 (2008) 380e386
Author's personal copy
[89]. The compound is currently in Phase II clinical trials for
allogeneic hematopoietic stem cell transplantation (http://
clinicaltrials.gov).
6. Conclusion and future directions
We have put forward a scheme (Fig. 1), which resolves the
apparent incompatibility of the dual role that cysteine cathep-
sins play in cancer progression: first by promoting tumour in-
vasion through ECM degradation, etc., and second by
participating in apoptotic pathways. Although these two path-
ways have apparently opposing functions, there is a clear
physical separation of the processes, enabling the possibility
of their selective targeting. Whereas the proteolysis required
for tumour invasion occurs mostly in the extracellular space,
the apoptosis pathway is clearly intracellular and dependent
on translocation of cathepsins into the cytosol. Therefore,
this differential distribution appears to be the key feature
that separates the two functions of cysteine cathepsins de-
scribed and provides an interesting and unique example how
the same enzymes can have such contradictory functions.
Given our current understanding of cathepsin activity in
tumour progression, it is evident that cysteine proteases could
be a rational target for cancer therapy. However, the pharmaceu-
tical approaches must differ, depending on the particular process
one would aim at. Consequently, while the use of a lysosomo-
tropic agents inducing lysosomal membrane permeabilization
could promote tumour cell death, the use of non cell-permeable
inhibitors of cathepsins would decrease the invasivepotential of
tumour cells in vivo. This underlines the need for further re-
search on the molecular mechanisms of the processes that cyste-
ine cathepsins are involved in, and on the development of
specific cathepsin inhibitors and lysosomotropic agents to gen-
erate new cancer therapies.
Acknowledgements
We apologize to the many contributors to this field whose
work we were unable to cite owing to space restrictions. We
would like to thank Dr Veronika Stoka for insightful discus-
sions and Prof. Roger H. Pain for critical reading of the man-
uscript. This work was supported by grants from the Ministry
of Higher Education, Science and Technology of the Republic
of Slovenia and from Human Frontiers Science Program
(RGP0024/2006-C).
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