Cysteine Cathepsin Proteases: Regulators of Cancer Progression and Therapeutic Response

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Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response

Oakley C. Olson1,2 and Johanna A. Joyce1,3,4 Abstract | Cysteine cathepsin protease activity is frequently dysregulated in the context of neoplastic transformation. Increased activity and aberrant localization of proteases within the tumour microenvironment have a potent role in driving cancer progression, proliferation, invasion and metastasis. Recent studies have also uncovered functions for cathepsins in the suppression of the response to therapeutic intervention in various malignancies. However, cathepsins can be either tumour promoting or tumour suppressive depending on the context, which emphasizes the importance of rigorous in vivo analyses to ascertain function. Here, we review the basic research and clinical findings that underlie the roles of cathepsins in cancer, and provide a roadmap for the rational integration of cathepsin-targeting agents into clinical treatment.

Extracellular matrix Our contemporary understanding of cysteine cathepsin tissue homeostasis. In fact, aberrant cathepsin activity (ECM). The ECM represents the proteases originates with their canonical role as degrada- is not unique to cancer and contributes to many disease multitude of proteins and tive enzymes of the lysosome. This view has expanded states — for example, osteoporosis and arthritis4, neuro macromolecules secreted by considerably over decades of research, both through an degenerative diseases5, cardiovascular disease6, obe- cells into the extracellular improved understanding of lysosomal biology and from sity7 and cystic fibrosis8, as well as parasitic9 and viral10 space, including collagens, laminins and fibronectin, which an increased appreciation of extra-lysosomal cathepsin pathogenesis. Cathepsins make crucial contributions to provide structural support as functions. Cysteine cathepsin proteases (BOX 1), referred tumour progression in numerous cancer types through a well as organizational to in this Review collectively as cathepsins, belong to variety of different mechanisms. Cathepsins are key acid architecture within the tissue. the papain superfamily of cysteine proteases, a class hydrolases within the lysosome, and represent the prin- Tumorigenesis is characterized 11,12 by remodelling of the ECM in of enzymes evolutionarily conserved as far back as cipal effectors of protein catabolism and autophagy , 1 association with disease the prokaryotes . In considering the role of cathepsins by which they support the increased metabolic needs progression. as lysosomal proteins, one must appreciate that the of proliferating cancer cells. Intracellular cathepsins are endolysosomal organelle system of higher eukaryotes also crucial components of signal transduction pathways evolved in parallel with these proteolytic enzymes. Thus that drive cancer cell growth and inflammation13–16. 1Cancer Biology and Genetics cathepsins are integrated into nearly all processes associ- Secreted cathepsins in particular have emerged as Program, Memorial Sloan ated with the lysosome: for example, protein degrada- potent effectors that modify the tumour microenvi- Kettering Cancer Center. 2Gerstner Sloan Kettering tion, protein and lipid metabolism, autophagy, antigen ronment through the turnover and degradation of the 17 Graduate School of Biomedical presentation, growth factor receptor recycling, cellular extracellular matrix (ECM) , and the activation, process- Science, Memorial Sloan stress signalling and lysosome-mediated cell death. In ing or degradation of various growth factors, cytokines Kettering Cancer Center, New a process termed lysosomal exocytosis, these enzymes and chemokines18,19. Cathepsins also shed cell–cell adhe- York, New York 10065, USA. 20–22 3Department of Oncology, can also be secreted, which results in the degradation sion molecules and contribute to tissue invasion and 20,21,23–27 University of Lausanne. of extracellular targets such as bone matrix and athero metastasis , and therefore represent an important 4Ludwig Institute for Cancer sclerotic plaques2,3. The broad range of biological pro- class of proteins that facilitate cancer progression and Research, University of cesses that utilize cathepsins indicates their importance dissemination. More recently, cathepsins have been Lausanne, CH‑1066 as crucial effector molecules. shown to be part of the dynamic response to anticancer Lausanne, Switzerland. Correspondence to J.A.J. To understand the roles of cathepsins in cancer it is therapy within the tumour microenvironment, and they e‑mail: [email protected] essential to fully dissect the complex processes by which can have crucial roles in the development of therapeutic doi:10.1038/nrc4027 dysregulation of proteolytic activity disrupts normal resistance14,28.

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Protease web In this Review we discuss the roles of individual human disease is summarized, and we describe how The interconnected system of cysteine cathepsins in cancer, and consider their unique mouse models of cancer have provided crucial insights biological interactions between functions as well as their integration within a protease into their various roles in malignancy. The focus on all proteases and protease network or protease web (BOX 2; FIG. 1). The utility of the mechanistic functions of cathepsin proteases inhibitors. cathepsins as prognostic and predictive biomarkers in compares and contrasts their normal roles with their

Box 1 | The cysteine cathepsin proteases In humans, the cysteine cathepsin family comprises 11 members (CTSB, CTSC, CTSF, CTSH, CTSK, CTSL (also known as CTSL1), CTSL2 (also known as CTSV) CTSO, CTSS, CTSW and CTSZ (also known as CTSX))143 (see the table). The majority of cysteine cathepsins are endopeptidases that cleave peptide bonds within their protein substrates. Additionally, CTSB possesses carboxypeptidase activity and CTSH possesses aminopeptidase activity. Two cysteine cathepsin family members do not have endopeptidase activity: CTSC and CTSZ. CTSC, also known as dipeptidyl peptidase I, is a strict dipeptidyl aminopeptidase, whereas CTSZ is a carboxymonopeptidase144,145. CTSZ has the unique feature of an integrin-binding Arg-Gly-Asp (RGD) motif in the pro-domain146. The enzymatically active site of cysteine cathepsins consists of a cysteine, a histidine and an asparagine residue that form a classic acid–base–nucleophile triad (CHN in the table). Additionally, the majority of these enzymes are further post-translationally modified by glycosylation, which is crucial for engagement with mannose‑6‑phosphate receptors (M6PRs) and sorting to the lysosome147. M6PRs can sort lysosomal hydrolases from the trans-Golgi network and recover secreted enzymes through the endocytic pathway. Indeed, the tumour-suppressive activity of M6PR is thought to be in part a result of its ability to suppress aberrant trafficking of lysosomal enzymes148. In the context of cancer, the dynamics of many of these processes are altered. Lysosomes transition from a perinuclear to a membrane-proximal position in response to oncogenic signalling53,149. These alterations in endolysosomal trafficking lead to the secretion of cathepsins into the extracellular space51,115. Infiltrating immune and stromal cells can be similarly activated to secrete cathepsin proteases into the extracellular space20,150, but the underlying mechanisms of this altered trafficking are poorly understood and remain an open question. As members of the C1 or papain family of proteases, cysteine cathepsins are synthesized as inactive zymogens, and cleavage of an inhibitory propeptide is required for full proteolytic activity. These propeptides are either cleaved by other proteases or autocatalytically removed at low pH. Multiple mechanisms have been identified by which propeptides either lose structure upon protonation or are excluded by stabilization of occluding loops, which ultimately results in exposure of the active site and subsequent autocatalytic cleavage151,152. These soluble propeptide fragments can retain their ability to inhibit cathepsin function and thus represent a further level of complexity in the regulation of cathepsin activity153,154. Cathepsin activity is optimized for the low pH and reducing environment of the lysosome113,155, which helps to restrict it to this compartment under normal conditions. However, it has long been appreciated that the extracellular environment of the tumour is acidified compared with normal tissue156,157. This adaptation supports the extracellular activity of secreted cathepsin proteases; also, low pH has been shown to induce the secretion of cathepsins158,159. Indeed, raising the pH of the microenvironment reduces invasion and metastasis in experimental models of carcinogenesis160.

Catalytic activity Subunit structure CTSB • Endopeptidase Dimer of heavy chain • Carboxypeptidase and light chain C H N CTSC Aminodipeptidase Tetramer of heterotrimers including exclusion C H N domain chain, heavy chain and light chain

CTSF Endopeptidase Monomer C H N

CTSH • Endopeptidase Trimer of heavy chain, Propeptide • Aminopeptidase light chain and mini chain C H N Exclusion domain chain CTSK Endopeptidase Monomer C H N Mini chain CTSL Dimer of heavy chain Light chain Endopeptidase C H N (CTSL1) and light chain Heavy chain CTSL2 Main chain Endopeptidase Monomer C H N (CTSV) CHN Catalytic triad CTSO Endopeptidase Monomer C H N RGD Integrin- binding motif CTSS Endopeptidase Monomer C H N N-linked glycosylation

CTSW Endopeptidase Monomer C H N

CTSZ Carboxymonopeptidase Monomer (CTSX) RGD C H N

100aa 200aa 300aa 400aa 500aa

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dysregulated activity within the tumour microenviron- of ~2,000 individuals showed a significant correlation ment. Ultimately, we dissect their functional complexity between elevated serum CTSS levels and increased and propose a framework for a meaningful understand- mortality risk in older adults38. Further analysis of these ing of the roles of cathepsins in cancer biology and cohorts revealed an association with both cancer mortal- integration of this knowledge into therapeutic strategies. ity and cardiovascular mortality38. A summary of these clinical studies is provided in TABLE 1. Cathepsins and clinical prognosis Given the myriad functions of cathepsins in cancer, It has long been recognized that cathepsin expression as discussed in this Review, it is important to critically is frequently increased in tumours in comparison with assess the relevance of these correlative studies to the normal tissue, and this was a major motivation for treatment of cancer patients. Specifically, it is essential to 29 N-terminomic and investigators to begin studying cathepsins in cancer . discriminate between the individual instances in which C‑terminomic approaches Increased cathepsin expression is significantly associ- cathepsin expression might provide important prognos- Covalent labelling of either the ated with poor prognosis in breast, lung, head and neck, tic information, or additionally indicate a therapeuti- N terminus or C terminus and colorectal cancer (CRC), melanoma and many other cally targetable weakness, and those patients for whom within a protein sample allows 30,31 (TABLE 1) identification of the original cancers . Cathepsins are additionally predic- it has no functional relevance. However, the assessment terminus in proteomic mass tive of response to anticancer therapy in several of these of aberrant cathepsin activity within the tumour is not spectrometry analysis. tumour types32–34. Expression of individual cathepsins always easily interpreted. The presence of endogenous can also discriminate between instances of venous ver- inhibitors of cathepsins, such as cystatins (FIG. 1), com- Cystatins 35 A protein family of cysteine sus lymphatic invasion , and correlates with site-specific plicates the simple use of expression alone as a surro- 21 39 protease inhibitors. metastasis . gate for protease activity . Conversely, assessment of Although analysis of resected tissue has been invalu- enzymatic activity from homogenized tissue samples is Tumour-associated able in establishing the utility of expression of cathepsin equally problematic as it disregards the cellular source, macrophages family members as important prognostic indices, the subcellular localization and intratumoural heterogene- (TAMs). These macrophages are ‘educated’ in the tumour measurement of circulating cathepsins in patient sera ity of cathepsin activity. Given these challenges, efforts microenvironment where has also emerged as a non-invasive means to generate have been made to measure cathepsin protease levels they provide various useful information on tumour malignancy. For instance, along with degradation of a known cathepsin substrate tumour-promoting functions. in CRC, high serum levels of cathepsin Z (CTSZ) were to indicate high levels of proteolytic activity40. Related TAMs have been shown to 36 promote tumour angiogenesis, associated with shorter overall survival , and CTSL lev- approaches include the development of activity-based 41–46 invasion and metastasis, els in the serum of patients with malignant ovarian can- probes for analysis of tumour tissue in situ (BOX 3). immunosuppression and cer were elevated compared with those who had benign Finally, the importance of cathepsins in tumour progres- therapeutic resistance. tumours or healthy controls37. Interestingly, a large study sion and patient outcome can often be ambiguous as to whether the cathepsins are mechanistically related to the reported phenotypes, are simply part of malignancy- Box 2 | The protease web associated transcriptional changes or are indicative of a The modern scientific paradigm of functional reductionism and the tools of genetics tumour-promoting immune cell infiltrate47. Therefore, and biochemistry have been instrumental in defining our understanding of the functional analyses have been crucial in moving beyond biological role of proteases. However, this intellectual framework is inherently limited correlative studies, as discussed below. as it biases our experimentation towards an explanation of phenotypes as the result of single proteases efficiently cleaving a limited set of substrates. The necessity for a given Cathepsins in the tumour microenvironment protease is delineated by genetic experiments or with chemical inhibitors. Sufficiency Tumours represent complex tissue microenvironments48, is determined through biochemical cleavage assays or by ectopic expression of the protease of interest. Although these approaches effectively define the capability of and in addition to cancer cells, many other cell types a protease, and the biological processes for which its activity is crucial, they can express cathepsins. These include fibroblasts, osteoclasts, overlook the nuance and extent of its full biological activity. However, adopting an neutrophils, mast cells, T cells, myoepithelial cells and integrated systems biology approach for the study of protease function seems to hold endothelial cells, although none expresses cathepsins as considerable promise for broadening our understanding. By defining the extensive robustly as tumour-associated macrophages (TAMs)20,23 mechanisms by which proteases alter the function of other proteases, through direct (BOX 3). Cathepsin expression is regulated in these processing as well as by alteration of protease inhibitors, recent research has created a various cell populations in a cell type-specific manner systems view of protease function and has allowed us to contemplate the global effect and their tumour-promoting or tumour-suppressive 161 of perturbation of a single protease . The relevant connection between the activities of functions can similarly vary. Expression of cathepsins distinct proteases is termed ‘reachability’ and represents a significant overhaul of our is directed by GC‑motif transcription factors SP1 and understanding of single protease–substrate pairs and linear protease cascades161. (REF 49) This analysis suggests potentially widespread misinterpretation of genetic and ETS1 and it is also part of a lysosomal biogenesis pharmacological data as the result of indirect effects on other proteases through their transcriptional network driven by the transcription fac- 50 reach into the protease web. Although this represents a substantial conceptual tor TFEB . In the context of the cancer cell, oncogene advancement with regard to protease activity, we still lag in our ability to understand activation lies upstream of induced cathepsin expression the subtle processing of substrates and aggregate phenotypes. As mass spectrometry in the majority of instances. For example, transformation approaches, particularly N-terminomic and C‑terminomic approaches162,163, become of mammary epithelial cells with mutant HRAS upregu- more widely used, it will hopefully be possible to determine where limited processing of lates both CTSB and CTSL51. CTSB is also trafficked to multiple substrates by a protease is responsible for a single phenotype. To this end, the the extracellular surface of the plasma membrane where term ‘degradomics’ has been introduced to refer to these proteomic and genomic it interacts with annexin 2 in caveolae51,52. Similarly, the approaches that aim to define the full complement of proteases and their substrates at HER2 (also known as ERBB2) oncogene drives expres- a tissue or organismal level164. sion of CTSB through the transcription factor myeloid

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Cystatin A, Serpin B13 B,3,5,6 or 7

Cystatin A, CTSZ CTSL CTSK CTSS B,3 or 5

Cystatin A, MMP2 uPA CTSD CTSC B,1 or 3

Mast cell tPA CTSB Elastase Granzyme A or B protease 2 or 4

Cystatin A, Cystatin A, MMP3 CTSG CTSH B,1,3 or 6 B,3 or 5

Cysteine protease Metalloproteinase Serine protease Aspartic protease Cystatin Serpin

Figure 1 | Integration of cysteine cathepsin proteases within a proteolytic network. VisualizingNature cathepsins Reviews within | Cancer their local network of proteases and inhibitors underscores the complexity of proteolytic activity in a given tissue environment. Unlike classic proteolytic cascades, many of these interactions are bidirectional and the overall network is nonlinear, which enables positive and negative feedback and compensation. Within the network, certain proteases such as cathepsin B (CTSB) and CTSL represent highly integrated nodes whereas others, such as CTSH, CTSK or CTSZ, represent more discrete activities. Note that the endogenous cathepsin inhibitors, cystatins and serpins, show a considerable level of molecular promiscuity and overlap in their specificity. Although not shown in this figure, this representative local network is embedded within the larger ‘protease web’ as coined by Overall and colleagues161,179 (BOX 2), and therefore further layers of complexity remain to be identified in addition to those shown here. MMP, matrix metalloproteinase; tPA, tissue plasminogen activator; uPA, urokinase-type plasminogen activator. Adapted with permission from REF. 180, Elsevier.

zinc finger 1 (MZF1), and CTSB is a functional driver of type specific, whether there are any post-translational RIP1‑Tag2 PanNET mouse 53 model the invasive phenotype . ETS transcription factors are modifications or biochemical differences in the pro- Expression of the SV40 large also crucial in regulation of the expression of CTSB and teases as a consequence of their cellular origin remains T antigen (Tag) under the CTSL in cancer cells49,54. Finally, interferon‑γ (IFNγ) has to be determined. control of the rat insulin been shown to induce CTSS expression in lung cancer The roles of individual cathepsins can vary dra- promoter (RIP) causes β-cell 55 hyperplasia, which progresses cells , which supports a role for tissue inflammation in matically between different tumour types. Studies of through a series of rate-limiting the regulation of cathepsin activity within the tumour CTSB and CTSC provide a particularly illuminating stages to invasive pancreatic microenvironment. example of this disease-specific variability. Although neuroendocrine tumour The education of macrophages within the tumour both cathepsins are upregulated during the course of (PanNET). microenvironment results in a pronounced upregula- tumorigenesis in multiple cancer types, the functional 24,58 MMTV-PyMT mammary tion of cathepsins. Interleukin‑4 (IL‑4) was identified as relevance of each is highly context dependent . CTSB carcinoma mouse model a key cytokine that induces cathepsin activity specifi- has potent tumour-promoting roles in the RIP1‑Tag2 Expression of the polyomavirus cally in TAMs20 (BOX 3). Although both cancer cells and PanNET mouse model24 and the MMTV-PyMT mammary middle T (PyMT) oncoprotein TAMs can produce certain cathepsins, it has become carcinoma mouse model26,59,60 (TABLE 2). Ctsb-deficient mice under control of the mouse mammary tumour virus clear that these two different cellular sources have a dif- have significantly reduced tumour volume, in associa- 24,26,59,60 (MMTV) long terminal repeat ferent impact on the tumour microenvironment. Bone tion with decreased cancer cell proliferation . By promoter induces mammary marrow transplantation strategies in a RIP1‑Tag2 PanNET contrast, Ctsb deletion has no effect on the progression neoplasia reminiscent of that mouse model demonstrated that TAM-derived CTSB, of the K14‑HPV16 mouse model of squamous cell carcinoma observed in human breast CTSH and CTSS were either predominantly or exclu- (SCC), despite abundant CTSB expression in both the cancer. MMTV-PyMT tumours 58 progress through stereotypical sively responsible for the tumour-promoting functions stromal and epithelial cell compartments . 20,56 stages of cancer progression of these distinct cathepsins (J.A.J. and V. Gocheva, Conversely, CTSC displays the opposite profile and metastasize to the lung. unpublished observations). Conversely, CTSL promoted of functional importance in tumour progression. pancreatic neuroendocrine tumour (PanNET) progres- Deficiency of this protease does not affect either K14‑HPV16 mouse model 20 24 58 of squamous cell carcinoma sion in a cancer cell-intrinsic manner , whereas CTSZ PanNET or mammary cancer progression in mice , Use of the keratin 14 promoter displayed tumour-promoting functions from both whereas in the context of SCC, Ctsc deletion results in (K14) to drive expression of cancer cell-derived and TAM-derived sources25. In an a potent block in disease progression58. Moreover, SCC human papilloma virus type 16 experimental CRC model, CTSS expression by both cells implanted orthotopically into Ctsc-null animals (HPV16) E6 and E7 genes in cancer cells and stromal cells was shown to be crucial fail to grow, implicating host-derived CTSC as a crucial basal cells of the squamous 57 epithelium results in stepwise for the promotion of CRC growth and progression . regulator of disease progression. Indeed, bone marrow progression to squamous cell Although these examples illustrate that the tumour- transplantation experiments and add-back of CTSC- carcinoma. promoting capacity of individual cathepsins can be cell expressing cancer-associated fibroblasts demonstrated

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specific contributions of leukocyte- and fibroblast- as JAM2)21, or to alternative mechanisms of tissue- derived CTSC in promoting angiogenesis and tumour specific proteolytic regulation. It is interesting to note growth. CTSC, which is a dipeptidyl aminopeptidase, is that the expression of CTSB and CTSC increases with capable of activating multiple proteases including gran- tumour progression, even in those models in which they zymes A and B, mast cell proteases 2 and 4, and neu- have no crucial function23,24,58. To this end, experiments trophil elastase61,62, which suggests that CTSC regulates to distinguish between the specificity resulting from the cancer progression through a central position within its cancer cell lineage of origin and that derived from local proteolytic network (FIG. 1). However, genetic defi- the tissue microenvironment have yet to be performed, ciency for either mast cell protease 4 (Mcpt4) or neutrophil and these will be crucial in discriminating between these elastase (Elane) had no significant effect on SCC tumour possible mechanistic explanations. progression58, which suggests the possibility that only In support of the concept that specific cathepsins the simultaneous loss of multiple downstream protease enhance tumorigenesis in certain tissue microenviron- targets will recapitulate the deficiency of Ctsc alone. ments, there is the example of the tumour-promoting role It remains unclear whether this differential tumour- of CTSB in both pancreatic ductal adenocarcinoma and KPC mouse model promoting activity of individual cathepsins in distinct PanNET. Genetic loss of Ctsb in pancreatic epithelial cells Addition of the LSL‑Trp53R172H KPC mouse model63 allele to the KC model, results organs is generally attributable to tissue-specific sub- in the and in neuroendocrine cells in 24 in widely metastatic invasive strates, as shown for the brain-enriched CTSS substrate the RIP1‑Tag2 mouse model , severely affects tumori- pancreatic ductal carcinoma. junctional adhesion molecule B (JAM‑B; also known genesis (TABLE 2). Despite the different cells of origin for

Table 1 | Cathepsins as prognostic and predictive factors in human cancer Disease Cathepsin expression Clinical association Ref. Breast cancer High CTSB and CTSL High CTSB and CTSL levels in the primary tumour are indicative of poor prognosis 21,32, for both disease-free survival and overall survival 33,67, 182–185 High CTSB and CTSL levels correlate with reduced hormone receptor immunoreactivity High CTSB High CTSB is associated with lymph node metastases in patients with inflammatory breast cancer and is a powerful prognostic indicator of disease recurrence and overall survival in patients with lymph node-negative disease High CTSL High CTSL is predictive of poor response to systemic adjuvant endocrine therapy in patients with hormone receptor-positive cancer High CTSB, CTSC, CTSL High CTSB, CTSC, CTSL and CTSS are predictive for brain-specific metastasis and CTSS CTSB, CTSC and CTSL Only CTSB, CTSC and CTSL are predictive for lung-specific metastasis Colorectal cancer High CTSB and CTSL High CTSB and CTSL levels are significantly correlated with disease metastasis 34, 186–188 High CTSB and CTSL activity is prognostic for poor overall survival in patients with colorectal cancer following curative resection High CTSL High levels of CTSL are associated with poor differentiation status High CTSS High CTSS expression is associated with reduced progression-free survival in patients treated with surgery alone. However, high CTSS expression was predictive for those patients who would derive benefit from adjuvant 5‑fluorouracil and folinic acid treatment Lung cancer High CTSB High CTSB expression is prognostic of shorter overall survival in both squamous cell 189–193 carcinomas and adenocarcinomas High CTSH High CTSH levels are associated with decreased survival only for the smoker patient population CTSS CTSS is positively associated with increased overall survival Ovarian cancer High CTSB High CTSB expression is a negative predictor of tumour debulking success. High 194 CTSB is negatively correlated with both progression-free survival and overall survival Pancreatic High CTSB and CTSL In resectable pancreatic adenocarcinomas, high CTSB and CTSL levels are 195 adenocarcinoma levels positively correlated with tumour grade and negatively correlated with overall survival following surgery High CTSB High CTSB is correlated with increased lymphatic invasion. High CTSB is also prognostic for recurrence within 6 months of surgical resection Pancreatic CTSB, CTSL and CTSZ Increased levels of CTSB, CTSL and CTSZ are positively associated with increased 24,25 neuroendocrine cancer tumour grade and invasion Osteosarcoma High CTSK High CTSK is positively associated with disease metastasis and poor overall survival 196 Illustrative examples of clinical findings for cathepsins as prognostic and predictive factors are provided here. The scope of this table has been restricted to malignancies that are discussed in the main text. For a more complete compendium of clinical associations please refer to REFS 30,31. CTS, cathepsin.

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each cancer type, loss of Ctsb results in decreased prolif- or vascular endothelial growth factor A (Veg fa) 69, two eration and reduced invasion and/or metastasis in both. crucial growth factors that are required for the rate- Interestingly, CTSB is also crucial for the induction of limiting steps of pancreatic neuroendocrine hyperplasia experimental pancreatitis, largely through its role in and angiogenesis, respectively. The tumour-promoting trypsinogen activation64. Thus, in these three pathologi- function of CTSL is mediated entirely by the cancer cal conditions of the pancreas, CTSB seems to be a crucial cell source of this enzyme, as the knockout phenotype disruptor of normal tissue homeostasis. cannot be rescued by transplantation of this mouse Although many cathepsins have been shown to model with wild-type donor bone marrow20. have either a tumour-promoting function or no effect However, the opposite effect is observed in the in specific cancer types (TABLE 2), CTSL is unique in skin microenvironment where loss of Ctsl enhances having the ability to either enhance or suppress car- carcinogenesis in both the genetic K14‑HPV16 cinogenesis in a context-dependent manner. CTSL is mouse model and the classic two-step chemi- often expressed at high levels in tumours; moreover, cally induced 7,12‑dimethyl-benz[a]anthracene– in some instances, particularly in breast and head and 12‑O-tetradecanoylphorbol‑13‑acetate (DMBA-TPA) neck cancer, CTSL expression serves as a valuable bio- mouse model of skin carcinogenesis15,70. In fact, Ctsl- marker with negative prognostic indication for time to knockout mice develop epidermal abnormalities such relapse and overall survival in patients65–67 (TABLE 1). In as keratinocyte hyperplasia71 that result from increased the context of the RIP1‑Tag2 PanNET mouse model, epidermal growth factor (EGF) recycling and deregu- CTSL has a potent tumour-promoting function, which lated autocrine mitogenic signalling15,72. These examples is the most pronounced of any of the cysteine cath- thus identify opposing, context-dependent functions epsins analysed20,24,25,56 (TABLE 2). Moreover, the deletion for CTSL in the regulation of cell proliferation as a of this protease in these mouse models is comparable to mechanistic explanation for the striking organ-specific genetic ablation of insulin-like growth factor 2 (Igf2)68 differences of this enzyme in cancer.

Box 3 | Activity-based probes The enzymatic activity of cathepsin proteases is tightly regulated at multiple levels: for example, proteolytic activation, pH, redox potential and stabilization via binding partners that include macromolecules such as the glycosaminoglycans of the glycocalyx. Additionally, there is a class of reversible but tight-binding endogenous inhibitors known as cystatins and serpins that provide a further level of control over proteolytic activity39. Moreover, cathepsin activity is restricted at the level of expression in a cell type-specific manner, by the subcellular and extracellular trafficking of the enzymes, and their activation, stabilization and inactivation by their immediate environment. The complexity of cathepsin activity regulation has necessitated the development of molecular probes that can label active cathepsins in situ. These activity-based probes (ABPs) use thiol-reactive electrophiles to covalently label the active site cysteine in an enzymatic activity-dependent manner. ABPs are useful not only for investigation into the role of cathepsins in the tumour microenvironment20,23 but also have applications in medical imaging and diagnostics42,46, as well as surgical guidance in the context of tumour resection43. Although ABPs can be very informative tools, interpretation of the data must always take into consideration the pharmacodynamics and pharmacokinetic properties of the ABPs. Cathepsins will only be labelled where the probes are bioavailable. Furthermore, the fact that ABPs are also inhibitors indicates that they are not necessarily biologically inert. An example of the use of ABPs in the analysis of PanNET progression is shown in the figure. The cathepsin ABP (CathABP) demonstrates the accumulation of cells with high cathepsin activity (red) in the progression from normal islets (N) to hyperplastic islets (H), angiogenic islets (A) and, finally, invasive tumours (T). Co‑staining with F4/80 (green) identifies these cells as macrophages. Intriguingly, the percentage of macrophages that are cathABP+ (indicated on each image) increases with tumour progression and is highly enriched at the invasive front where the tumour invades the surrounding normal exocrine (E) tissue. DAPI, 4',6'-diamidino-2-phenylindole. Images are reprinted with permission from REF. 20, CSH Press.

Normal Hyperplastic (8 weeks) Angiogenic (10 weeks) Tumour (13.5 weeks) A E

E H N E A T E

A E

E H N E A T

0% 5±2% 69±9% 85±3% DAPI CathABP F4/80

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Angiogenic switching These genetic experiments have been instrumental Deletion of Ctsb in MMTV-PyMT mice resulted in The stage at which incipient in elucidating the regulatory functions of cathepsins in increased CTSZ at the surface of cancer cells, which tumours acquire the capacity cancer, and in contributing to our understanding of was proposed to mask phenotypes of CTSB deficiency to induce angiogenesis. the underlying molecular mechanisms, as is discussed in this mouse model26,59. Indeed, combined ablation of further below. In addition, analyses in genetically engi- Ctsb and Ctsz resulted in pronounced decreases in both neered mouse models have revealed both the functional primary tumour development and lung metastasis59 redundancy of individual cathepsins, and the potential (TABLE 2). In the RIP1‑Tag2 mouse model, combined for compensation from other family members when mul- deletion of Ctsb and Ctss resulted in an additive effect tiple nodes in the protease web are perturbed. Two illus- in the reduction of angiogenic switching in preneoplastic trative examples of these phenomena were uncovered lesions (J.A.J., L. Akkari, V. Gocheva et al., unpublished in the MMTV-PyMT and RIP1‑Tag2 mouse models. observations). However, later in tumour progression,

Table 2 | Genetic studies of cathepsin and cystatin functions using genetically engineered mouse models of cancer Gene Cancer type Genetically engineered Phenotype Refs knockout mouse model Cysteine cathepsins Ctsb−/− PanIN LSL-KrasG12D; PDX1‑Cre Delay in disease onset and 54% reduction in proliferation of 63 PanINs; no difference in apoptosis PDA LSL-KrasG12D; LSL‑Trp53R172H; Delay in disease onset and 23% reduction in proliferation of 63 PDX1‑Cre PDA; 30% reduction in incidence of liver metastasis PanNET RIP1‑Tag2 72% reduction in tumour burden; significant decrease in 24 tumour invasion; 229% increase in apoptosis, 44% reduction in proliferation and 56% decrease in angiogenesis Mammary adenocarcinoma MMTV-PyMT 40% reduction in tumour burden (by weight) and 50% 26,60 reduction in lung metastasis; reduced cell proliferation Squamous cell carcinoma K14‑HPV16 No effect on tumour progression or tumour grade 58 Ctsc−/− PanNET RIP1‑Tag2 No effect on tumour progression 24 Mammary adenocarcinoma MMTV-PyMT No effect on tumour progression 58 Squamous cell carcinoma K14‑HPV16 Decreased tumour progression and tumour grade 58 Ctsh−/− PanNET RIP1‑Tag2 40% reduction in tumour burden; 2‑fold increase in 56 apoptosis and 59% decrease in angiogenesis Ctsl−/− Squamous cell carcinoma K14‑HPV16 Increased tumour progression with increased tumour 15 grade; 180% increase in lymph node metastasis PanNET RIP1‑Tag2 88% reduction in tumour burden; 337% increase in 24 apoptosis and 58% decrease in proliferation Ctss−/− PanNET RIP1‑Tag2 47% reduction in tumour burden; 164% increase in 24,103 apoptosis and 42% decrease in angiogenesis Ctsz−/− Mammary adenocarcinoma MMTV-PyMT No effect on tumour burden or proliferation; reduced 59 tumour cell death PanNET RIP1‑Tag2 63% reduction in tumour burden; 1.8‑fold increase in 25 apoptosis and 86% decrease in proliferation; no effect on angiogenesis Ctsb−/−Ctsz−/− Mammary adenocarcinoma MMTV-PyMT 45% reduction in tumour burden; no difference in cell 59 proliferation Ctsb−/−Ctss−/− PanNET RIP1‑Tag2 51% reduction in tumour burden; 85% decrease in * proliferation; no effect on angiogenesis or apoptosis Ctsb−/−Ctss−/− PanNET RIP1‑Tag2 58% reduction in tumour burden; 3.8-fold increase in * Ctsz−/− apoptosis and 53% decrease in proliferation; 73% decrease in tumour vascularization Cystatins Cstb−/− Mammary adenocarcinoma MMTV-PyMT 23% reduction in primary tumour by weight; no effect on 197 metastasis Cst3−/− Squamous cell carcinoma K14‑HPV16 Increased tumour progression or angiogenesis 198 PanNET RIP1‑Tag2 Increased tumour burden with increased tumour 103 proliferation and increased angiogenesis Cst, cystatin; Cts, cathepsin; HPV16, human papilloma virus type 16; K14, keratin 14 promoter; MMTV, mouse mammary tumor virus; PanIN, pancreatic intraepithelial neoplasia; PanNET, pancreatic neuroendocrine tumour; PDA, pancreatic ductal adenocarcinoma; PDX1, pancreas and duodenum homeobox 1; PyMT, polyoma middle T antigen; RIP1, rat insulin promoter; Tag2, SV40 large T antigen. *J.A.J., L. Akkari and V. Gocheva, unpublished observations.

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CTSZ is specifically upregulated and is associated with fibroblasts display enlarged autolysosomes, which indi- a reversal of several tumorigenic phenotypes, which sug- cates impaired catabolism of the cargo and decreased gests functional compensation by this family member. autophagic flux12. Another study examining angioten- Consistent with this hypothesis, deletion of Ctsz in a sin II (ANGII)-induced autophagy in macrophages Ctsb−/−Ctss−/− mutant background resulted in a substan- reported that CTSS deficiency resulted in autophago- tial block in tumorigenesis in RIP1‑Tag2 mice (J.A.J., L. some accumulation, which supports a role for CTSS in Akkari, V. Gocheva et al., unpublished observations) the fusion of the autophagosome with the lysosome11. (TABLE 2). Insights from these genetic studies could also Thus, cathepsins can have roles as both effectors and have important implications for the therapeutic targeting regulators of autophagic catabolism. of cathepsins using pharmacological agents, and suggest In cancer initiation, it is proposed that autophagy that pan-cathepsin inhibitors may be more efficacious suppresses transformation in response to oncogene in the long term than selective inhibitors of individual activation74, yet this can ultimately be overridden. The cathepsins, as discussed further below. nutrient sensor mTOR is crucially important for licensing cell growth driven by oncogenic PI3K–AKT signalling Cathepsin functions in lysosome biology and for suppressing autophagy. Thus, the lysosome and Cathepsins and protein catabolism in the lysosome. One its role in amino acid recycling contribute to the enable- of the major functions of the lysosome, and the proteases ment of cancer proliferation. Indeed, mTOR is regulated it contains within its acidic lumen, is the catabolism of by the Ragulator complex, which assesses free amino acid proteins and degradation into their amino acid com- content within the lysosomal lumen by measuring the ponents73 (FIG. 2). Proteins can traffic to the lysosome efflux of specific amino acids across the lysosomal mem- via the endocytic pathway or they can be sequestered brane75–77. Moreover, pancreatic ductal adenocarcinoma from the cytosol in a process known as autophagy by cells have been shown to be nutrient poor and dependent which the autophagosome, a double-membraned vesi- on macropinocytosis of extracellular protein as an amino cle that contains the protein cargo, fuses with the lyso- acid source78,79. Increased activity of the microphthalmia/ some. The protein cargo is then degraded by cysteine transcription factor E (MiT/TFE) family of transcrip- cathepsins and other lysosomal proteases in the newly tion factors is responsible for metabolic reprogramming formed autolysosome. Ctsl-deficient mouse embryonic of pancreatic cancer through increased expression of

Cell survival ECM Cell death Integrin α Integrin β Macropinocytosis P of extracellular RGD SRC proteins FAK CTSZ P Apoptosis

Endosome Cell survival and Lysosome proliferation MOMP CTSS Lysosome CTSL CTSB CTSS Mitochondrion Free CTSL amino mTOR LMP BID tBID CTSB acids

MiT/TFE-dependent lysosomal biogenesis ROS or lipid peroxidation

Autophagolysosome Autophagolysosome Autophagic cell death

Figure 2 | The yin and yang of cancer-associated lysosomal biogenesis. membrane integrity is compromised, either partiallyNature or completely, Reviews | Cancer in a The increased lysosomal biogenesis and expression of cathepsins in process termed lysosomal membrane permeabilization (LMP). Release of cancer cells both supports and endangers the survival of those cells. cathepsins into the cytosol can lead to mitochondrial outer membrane Increased catabolic activity is crucial for provision of the necessary permeabilization (MOMP) and apoptosis through the cleavage of nutrients to license cell proliferation82. Additionally, cathepsin Z (CTSZ) BH3‑interacting domain death agonist (BID) (which produces truncated has a crucial non-catalytic function in promoting increased focal BID (tBID)) by CTSB or CTSL93. ECM, extracellular matrix; MiT/TFE, adhesion kinase (FAK) and SRC signalling and proliferation in a cancer microphthalmia/transcription factor E; RGD, Arg-Gly-Asp; ROS, reactive cell-intrinsic manner25. In conditions of stress, however, lysosomal oxygen species.

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REVIEWS

lysosomal genes such as CTSS80. This increased lysosomal does LMP and cathepsin release promote apoptosis, it and autophagic activity is crucial for pancreatic tumori also actively suppresses survival signalling and alternative genesis as it allows cancer cells to capitalize on alternative forms of cell death. For example, CTSB released into the sources of amino acids. In conditions of essential amino cytosol can cleave the lipid signalling enzyme sphingo- acid scarcity, lysosomal degradation of internalized pro- sine kinase 1, which protects against sphingosine- and teins reactivates mTOR, which in turn suppresses lyso- ceramide-induced cell death95. CTSB and CTSS have somal catabolism and, paradoxically, cell proliferation81. been shown to degrade receptor-interacting Ser/Thr pro- Lysosomal protein catabolism is thus a necessary regu- tein kinase 1 (RIPK1), thereby suppressing the necropto- lator of cancer cell proliferation and a crucial survival sis cell death pathway and instead enforcing apoptosis96. pathway in the nutrient-stressed cell. In addition to depolarizing mitochondria, activated pro-apoptotic regulators BAX and BAK are capable Lysosomal membrane permeabilization and cathepsins of permeabilizing the lysosomal membrane97. The role of as effectors of cell death. Transformed cells express LMP in the amplification of apoptotic signalling is par- higher levels of lysosomal proteins and it has been sug- ticularly important in cytokine-mediated cell death, and gested that this reliance on lysosomal content could in Ctsb-deficient hepatocytes the ability of tumour necro- represent an ‘Achilles heel’ by which cancer cells might sis factor (TNF) to induce death is severely impaired98. be targeted to trigger their own death82. Indeed, trans- Effective mitochondrial membrane depolarization and formed cells are sensitized to lysosomotropic agents release of cytochrome c downstream of caspase 8 activa- capable of inducing lysosomal membrane permeabilization requires BID-dependent LMP99. However, LMP also tion (LMP) and subsequent programmed cell death83–85. operates as an independent pathway because even in cells Conversely, the survival factor heat shock protein 70 in which apoptosis has been compromised through the (HSP70) supports the integrity of the lysosomal mem- loss of crucial effectors such as BAX, BAD or caspases, brane and suppresses LMP, and has been shown to be LMP still leads to cell death100,101. Indeed, although the crucial for cancer cell survival86,87. apoptotic pathway is generally deregulated in cancer LMP can occur either in a limited manner that the obligate metabolic requirement for robust lysosomal allows discrete amounts of cathepsins and other lyso- activity described above indicates that the LMP pathway somal hydrolases access to the cytosol, or in a more of programmed cell death is probably generally intact. extensive manner that leads to a committed lysosome- mediated cell death. Lysosomes are acute sensors of Functions of extra-lysosomal cathepsins oxidative stress within the cell, and lipid peroxidation It is not always the case that extra-lysosomal cathepsins leads to impaired barrier function of the lysosomal in the cytosol are the result of LMP and are indicative of membrane, which results in LMP88. Dysregulation impending cell death. Although it remains unclear how of the lipid composition of the lysosomal membrane these proteases access the cytosol, CTSZ and CTSH have can also lead to LMP. This can occur as a result of an been shown to modulate immune cell–ECM attachment increase in the enzymatic activities of phospholipase via proteolytic modulation of cytoplasmic signalling A2 and phospholipase C89, or from aberrant acid pathways (BOX 4). Recent work has provided additional sphingomyelinase activity85. An intriguing physiologi- insight into the role of CTSZ in promoting ECM adhe- cal example of lysosomal lipid dysregulation that leads sion and proliferation in cancer. CTSZ-deficient PanNET to LMP and cathepsin-dependent cell death occurs in cancer cells show impaired proliferation and decreased the involuting mammary gland. Activation of signal adhesion to ECM proteins; for example, fibronectin, transducer and activator of transcription 3 (STAT3) collagen and the reconstituted ECM mixture Matrigel25. in mammary epithelial cells not only upregulates the CTSZ is crucial for active focal adhesion kinase (FAK) expression of CTSB and CTSL and downregulates their and SRC signalling within cancer cells in a catalytically endogenous inhibitor serpin 2A (SPI2A; also known as independent manner; the signalling relies instead on SERPINA3G) but also abrogates the secretory pheno the Arg-Gly-Asp (RGD) motif uniquely found within the type of these cells, leading to the accumulation of milk propeptide of CTSZ (BOX 1). Paradoxically, extracellular fat globules in lysosomal vacuoles90,91. STAT3 activa- supplementation of full-length CTSZ is unable to rescue tion causes phagocytosis of secreted milk fat glob- the proliferative defects of Ctsz-null cancer cells, which ules by mammary epithelial cells and the resultant indicates that its growth-promoting activity is entirely increase in lysosomal free fatty acid results in LMP and cytoplasmic. CTSZ has been reported to colocalize with cell death91. integrins in membrane-proximal vesicles102 and it is When LMP occurs in response to cellular stress, the probably in these vesicles, rather than on the cell sur- release of active cathepsins initiates mitochondrial cell face, that the CTSZ RGD motif exerts its pro-prolifera- 92 (FIG. 2) Apoptosome death programmes . This can occur through tive function. The emergence of catalysis-independent A pro-apoptotic structure the cleavage of BH3‑interacting domain death agonist roles for cathepsins in tumour progression represents an comprising cytochrome c, (BID) by cathepsins, resulting in cytochrome c release exciting advance in the field and it will be of interest to apoptotic protease-activating and activation of the apoptosome93. Cathepsins also cleave determine whether other cysteine cathepsins have simi- factor 1 (APAF1) and caspase the mitochondrial-resident anti-apoptotic molecules lar non-proteolytic functions. Together, these findings 9 formed upon mitochondrial outer membrane BCL‑2, BCL‑Xl and myeloid cell leukaemia 1 (MCL1) demonstrate that important moonlighting roles exist for permeabilization and release of and thus promote apoptosis by reducing the threshold cathepsins independently of their typical lysosomal and cytochrome c into the cytosol. for mitochondrial membrane depolarization94. Not only extracellular locations.

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Cathepsins in the tumour microenvironment MMPs in turn degrade cystatin E, cystatin C and cystatin Cathepsins regulate angiogenesis. Various studies have M, thereby increasing cathepsin activity107. CTSL is cru- implicated cathepsins in the regulation of tumour angio cial for the activation of heparanase108, which in turn can genesis20,24,56,57,103 (TABLE 2). CTSS can stimulate angiogenesis modulate angiogenesis and lymphangiogenesis109 through directly by generating pro-angiogenic peptide fragments the controlled release of heparan sulfate-bound growth from the ECM protein laminin 5 (REF. 103). Conversely, factors. CTSL is also necessary for neovascularization by CTSL and CTSS also generate the anti-angiogenic peptide promoting the invasion of endothelial progenitor cells endostatin from collagen XVIII104, thus demonstrating into the stroma and their subsequent incorporation into the complex nature of these proteases in the regulation of blood vessels110. CTSK is upregulated in direct response angiogenesis in the tumour microenvironment. Cathepsins to hypoxia and increases the proteolytic activation of have also been shown to lie downstream of VEGFA sig- NOTCH1 in endothelial cells, thereby inducing neovascu- nalling, wherein VEGFA leads to an altered ratio of cath- larization in response to ischaemic injury111. Collectively, epsins to their endogenous inhibitors, which results in an these studies demonstrate that not only are cathepsins overall increase in cathepsin activity. Deletion or inhibi- crucial regulators of the ECM remodelling required for tion of CTSB demonstrated its importance in basement angiogenesis and neovascularization, but they are also membrane degradation for the generation and expansion involved in modulation of the signalling pathways that of new ‘mother vessels’ within tumours105. In addition drive these processes. to their roles as direct effectors of angiogenic signalling, cathepsins can also regulate pro-angiogenic proteolytic Cathepsins are involved in cell–cell junction disruption, networks. Degradation of the endogenous tissue inhibi- cancer cell invasion and extravasation. To date, the tors of metalloproteinases (TIMPs) by CTSB increases the main focus of studies on the contribution of proteases to pro-angiogenic functions of matrix metalloproteinases tumour invasion has been on their role in ECM degrada- (MMPs) within the tumour microenvironment without tion and the establishment of invasion tracks. However, the requirement for any change in MMP expression106. it is becoming increasingly evident that proteases have

Box 4 | Cathepsins in immune cell function In order to understand the specific functions of cysteine cathepsins in the infiltration of immune cells, including in cancer, one must appreciate the crucial role of lysosomal-mediated protein degradation in antigen presentation. The function of an antigen-presenting cell is to sample internalized proteins through the generation of peptide fragments by proteolytic cleavage and to subsequently display these antigens to cells of the adaptive immune system. Thus it is not surprising that any proteolytic processing event that occurs in the lysosome will rely heavily upon cathepsins. Intriguingly, not only are the antigens themselves cleaved into smaller peptides suitable for presentation by cathepsins165, but the antigen machinery itself is activated, processed and regulated by these very same proteases. After major histocompatibility complex class II (MHC II) complexes are transcribed and assembled in the endoplasmic reticulum (ER), their molecular chaperone, the invariant chain (Ii), must be degraded before loading the peptide. In a mechanism that prevents premature loading of the complex, Ii is proteolytically processed in the lysosome by cathepsins. Cathepsin L (CTSL) and CTSS have non-redundant roles in the degradation of Ii following initial processing by asparagine endopeptidase121,166–168. Interestingly, whereas thymic epithelial cells rely upon CTSL for Ii chain degradation, in macrophages CTSF has an important role169. This can be viewed not necessarily as an example of redundant functions for cathepsins, but rather as an instance of how the diversity in molecular and biochemical character of the different cathepsins is used to generate biological diversity across distinct cell types. Thus, differential endolysosomal proteolytic function yields unique characteristics in antigen presentation. This concept of cathepsins as integral regulators of lysosomal function rather than simply degradative enzymes is further supported by the example of CD1d‑restricted antigen presentation. The efficient presentation of glycolipids by CD1d‑expressing thymocytes requires CTSL enzymatic activity170. Although the CTSL substrate in this context remains to be identified, this demonstrates an integration of cathepsins in myriad lysosomal functions above and beyond simple protein catabolism. Further examination of the endolysosomal system in immune cells reveals that cathepsins also have crucial roles in secretory granule function. Natural killer (NK) cells depend on CTSC activity for activation of granzyme B and their cytolytic function171, and other immune cells depend on CTSH to perform the same function172. Similarly, CTSL is involved in the processing of perforin173. Intriguingly it has been demonstrated that during the process of lymphocyte degranulation, CTSB is released and attaches to the plasma membrane, thereby protecting the cell from released cytotoxic effector molecules174. Immune cells also rely on cathepsins for proteolytic degradation of extracellular matrix (ECM) proteins in order to migrate and invade through tissue175, and this mechanism can be co‑opted to enhance cancer cell migration20. By contrast, CTSZ has emerged as a crucial regulator of lymphocyte migration in a manner that is independent of ECM degradation. The carboxypeptidase has been found to localize in the cytosol of lymphocytes where it can process the cytoplasmic tails of integrins, to promote their activation and cellular migration176,177. In a similar manner, CTSH can process the integrin and cytoskeletal integrator talin, which leads to an increase in the invasive behaviour of cells178. Interestingly, macrophage- secreted CTSZ can be transferred to the surface of cancer cells, where it enhances their motility in an integrin-dependent manner25. In sum, cathepsins are involved in immune cell function in myriad ways. In the context of cancer biology, it will be important to consider the possibility that the increased expression of cathepsins by cancer cells may represent a form of ‘leukocytic mimicry’ by which they develop a more invasive and immune-privileged behaviour21.

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a CTSL CTSB Fibroblast CTSS TAM Non-invasive tumour cell Pro-angiogenic fragments T cell

Cathepsin secretion into the extracellular space by TAMs and degradation of the ECM

MDSC b ECM

Highly metastatic tumour cell E-cadherin c CTSZ Integrin α RGD CTSB, CTSS and CTSL cleave E-cadherin RGD and disrupt tumour cell–cell junctions

Integrin β Invading tumour cell

RGD-mediated binding of secreted CTSZ to cancer cell integrins promotes tumour invasion

d Blood–brain JAM-B barrier

Brain microvasculature

Brain parenchyma

CTSS promotes cancer cell extravasation in the CNS through cleavage of JAM-B

Figure 3 | Cathepsin proteases in tumour progression and the metastatic cascade. a | CathepsinsNature canReviews be supplied | Cancer from multiple cellular sources within the tumour microenvironment, including cancer cells and infiltrating immune cells such as tumour-associated macrophages (TAMs)20. Cathepsins have crucial roles both intracellularly and extracellularly in the promotion of tumour progression, for example, by extracellular matrix (ECM) degradation. b | Secreted cathepsin B (CTSB), CTSL and CTSS can cleave the cell adhesion molecule E‑cadherin, promoting cancer cell invasion into the surrounding tissue24. c | The pro-form of CTSZ, secreted by either TAMs or cancer cells, has been shown to bind to cancer cell integrins through the Arg-Gly-Asp (RGD) domain to promote invasion25. d | Secretion of CTSS by circulating breast cancer cells has been shown to be crucial for their ability to cross the blood–brain barrier and metastasize to the central nervous system (CNS). Cancer cells use this proteolytic activity to cleave junctional adhesion molecules, specifically JAM‑B, in order to disrupt the integrity of the blood–brain barrier and allow for their extravasation21. MDSC, myeloid-derived suppressor cell. Adapted from REF. 181, Olson, O. C. & Joyce, J. A. A splicing twist on metastasis. Sci. Transl Med. 5, 169fs2 (2013) Reprinted with permission from AAAS.

important additional specialized functions: for example, Specific cleavage of cell–cell adhesion molecules has the cleavage of cell–cell adhesion molecules in order to begun to emerge as an important mechanism of tumour free cancer cells from their neighbours, which promotes promotion by cathepsins secreted into the extracellu- a highly invasive phenotype112 (FIG. 3). E‑cadherin is a cell lar space. Indeed, CTSS-mediated adhesion molecule adhesion molecule and an important epithelial tumour shedding has recently been reported at a later step in suppressor owing to its ability to both maintain tissue the metastatic cascade. In the context of breast-to‑brain organization and suppress intracellular signalling. It has metastasis, CTSS mediates cleavage of several JAMs, been identified as a substrate for CTSB, CTSL and CTSS most notably JAM‑B, to allow cancer cells to breach the Blood–brain barrier in RIP1‑Tag2 tumours24. The activity of these proteases blood–brain barrier21 (FIG. 3). Crucially, genetic or pharma- A highly selective vascular barrier between the circulation at the invasive edge of the tumour is associated with cological perturbation of CTSS activity hindered the abil- and the extracellular fluid of E‑cadherin loss from the cell surface and disaggregation ity of metastatic cells to effectively seed and colonize the the brain. of the tumour. brain. It is important to note that among the cathepsin

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family members, CTSS is best able to retain its optimal CTSS was identified as part of a gene signature specific proteolytic activity profile at neutral pH113. Outside the to the ionizing radiation-induced tumours117. Here, acidic pericellular environment of the tumour, many of radiation-induced upregulation of CTSS was also of func- the other cathepsins probably display reduced activity, tional consequence, as shown by promoting enhanced whereas CTSS retains its functionality even in a neutral transformation of normal mammary epithelium cells pH environment such as the microvasculature of the grown in soft agar117. brain. This further emphasizes the importance of tis- Whereas cancer cell-intrinsic upregulation of cath- sue context and microenvironment with regard to the epsins occurs in a lineage- and therapy-specific manner, specificity and redundancy of individual cathepsins in a more general mechanism has emerged whereby chemo their tumour-promoting functions. Recently, broader therapy treatment results in increased accumulation of sheddase functions for CTSB, CTSL and CTSS have macrophages that express high levels of cathepsins28. been reported that result in the cleavage of cell adhesion It has been demonstrated that orthotopic injection molecules, transmembrane proteins and other molecules of necrotic cancer cells alone is sufficient to drive the from the cancer cell surface22, thus potentially further recruitment of myeloid cells118, which supports the idea contributing to tumour invasion. that the increase in myeloid cells in response to thera- Many of the pro-invasive functions of cathepsins peutic treatment bears many similarities to a traditional necessitate their altered trafficking either to the cell wound healing response. This inflammatory process surface or into the extracellular space, as recently ultimately results in increased cathepsin protease activ- reviewed112. Interestingly, JAMs are targeted to the lyso- ity within the tumour microenvironment after chemo- some and degraded by cathepsins during transforming therapeutic treatment, and it has been observed in both growth factor‑β (TGFβ)-induced epithelial to mesenchy- mouse models and clinical samples from patients with mal transition (EMT) in breast cancer cells114. The same breast cancer28. proteolytic cleavage event can occur by regulated traf- In considering the increasing evidence for contribu- ficking of the substrate to the protease or alternatively tions from the reactive stroma and cathepsins in the devel- through relocalization of the protease to the substrate. opment of acquired chemoresistance, the potential benefit This inversion of the typical trafficking process demon- of including cathepsin inhibitors in therapeutic regimens strates how inexorably linked the roles of intracellular is a logical inference. TAMs protect breast cancer cells and extracellular cathepsin proteases can be. Another from chemotherapy-induced cell death in a cathepsin- intriguing mechanism by which cathepsins can be mis- dependent manner28. Experiments using macrophages routed within the cell, and either secreted or recycled, is from different cathepsin-null mice identified CTSB via lineage-specific wiring of the endolysosomal path- and CTSS as key mediators of this chemoprotective way in melanoma115. This occurs in a RAB7‑dependent effect. Additionally, use of the pan-cathepsin inhibitor manner, with RAB7 knockdown leading to enhanced JPM-OEt suppressed this microenvironmental adapta- secretion of multiple lysosomal cathepsins and matrix tion to therapeutic treatment and improved response proteins, and to a consequent increase in melanoma to chemotherapy28. This was similarly observed in invasion. a mouse model of pancreatic ductal adenocarcinoma in which gemcitabine was administered in combina- Cathepsins and therapeutic resistance tion with the pan-cathepsin inhibitor E-64 (REF. 63). The Damage to the tumour is an integral objective in the combination of the two agents doubled median survival majority of therapeutic interventions. Implicit in this to 23 days, compared with 11 days for the cohort treated process, however, is the reactive stress response trig- with vehicle only. Although not statistically significant, gered in the residual cancer cells and stroma. Many can- it was an improvement over treatment with gemcitabine cer and stromal cells become activated and mount an alone for which the median survival was only 14 days63. inflammatory and wound healing response that involves Additionally, CTSS levels in various CRC cell lines and increased proteolytic activity, which augments tumour endothelial cells increased following treatment with regrowth and can lead to acquired therapeutic resistance. irinotecan (also known as CPT‑11). Consequently, an Increased cathepsin levels after treatment have been enhanced response to CPT‑11 in combination with reported in several studies, underlining their impor- CTSS inhibition was observed in a CRC xenograft tance in this reactive and adaptive process28,116 (FIG. 4). model119. In each of these examples the cathepsin inhib- This can occur entirely in a cancer cell-intrinsic man- itor had no effect on tumour progression as a mono- ner as described for breast cancer cells in response to therapy, implying that the stress of chemotherapeutic ionizing radiation116. In this instance, ionizing radiation- treatment unmasked a reliance on cathepsins. induced DNA damage caused IFNγ induction and, Further evidence also demonstrates the importance ultimately, through the downstream transcription fac- of cathepsins in chemotherapy-induced inflammation. tor interferon regulatory factor 1 (IRF1), increased Myeloid-derived suppressor cells (MDSCs) are a subset expression of CTSS. Moreover, increased CTSS levels of infiltrating immune cells that are particularly sensi- Myeloid-derived suppressor resulted in decreased responsiveness to ionizing radia- tive to chemotherapeutic treatment. Gemcitabine and cells tion, demonstrating a role for this protease in cancer 5‑fluorouracil (5‑FU) have been shown to be espe- Tumour-associated myeloid cells that are phenotypically cell-intrinsic acquired resistance to ionizing radiation. cially effective at depleting these cells from the tumour 14 characterized by their ability to In a study comparing rat mammary tumours induced microenvironment . However, during the process of suppress T cell function. by ionizing radiation with those induced by carcinogen, chemotherapy-induced cell death, CTSB is released from

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a Intrinsic resistance Fibroblast TAM CD4+ MDSC ECM IL-1β T cell IL-17 • Gemcitabine IL-17 drives tumour • 5-FU regrowth and limits response to 5-FU and gemcitabine Tumour cell CTSB-dependent IL-1β drives Apoptotic production of IL-1β by production of IL-17 cell Invasive the NLRP3 inﬂammasome in CD4+ T cells tumour cell b Adaptive resistance IFNγ CTSS

IRF1 Upregulation Radiation of CTSS in CTSS cancer cells

Increased intratumour IFNγ following radiation c Inﬂammatory DAMPs cytokines Cathepsins • Paclitaxel • Etoposide

Monocyte

Release of DAMPs Increased inﬁltration Increased levels of active cathepsin and inﬂammatory of TAMs from the proteases in the tumour microenvironment cytokines by circulation following following treatment interfere with apoptotic cells therapeutic treatment chemotherapy-induced cell death

Figure 4 | Cathepsin proteases and therapeutic resistance. Cathepsins have been shown to be crucial regulators of the therapeutic response that leads to microenvironment-mediated therapeutic resistance. This canNature occur either Reviews through | Cancer intrinsic resistance, whereby cathepsin activity within the naive tumour microenvironment is sufficient to mediate survival signals, or through adaptive mechanisms whereby the therapeutic insult triggers a reactive increase in cathepsin activity within the tumour microenvironment. a | In the case of intrinsic resistance it has been observed that the antimetabolites gemcitabine and 5‑fluorouracil (5‑FU) cause lysosomal membrane permeabilization in intratumoural myeloid-derived suppressor cells (MDSCs) and subsequent cathepsin B (CTSB)-dependent inflammasome activation. The subsequent activation of the interleukin‑1β (IL‑1β) signalling cascade within the tumour microenvironment compromises therapeutic efficacy14. b | In the context of ionizing radiation, cancer cells have been shown to adapt to treatment by upregulating CTSS in an interferon‑γ (IFNγ)-dependent manner116. c | The adaptive upregulation of cathepsins can also occur through the increased recruitment of cathepsin-high tumour-associated macrophages (TAMs) in response to chemotherapeutic agents such as paclitaxel or etoposide28. These adaptive increases in intratumoural cathepsin activity levels blunt therapeutic efficacy, which can accordingly be improved by cathepsin inhibition. DAMPs, danger-associated molecular patterns; IRF1, interferon regulatory factor 1; NLRP3, NOD-like receptor family, pyrin domain containing 3.

the lysosomes of MDSCs, which results in inflammasome suppresses the production of IL‑1β and the severity of activation and IL‑1β production. IL‑1β in turn stimulates chemotherapy-induced intestinal mucositis120. This sug- CD4+ T cells to produce IL‑17, the pro-angiogenic effects gests that cathepsin inhibition might not only improve of which limit the efficacy of gemcitabine and 5‑FU in therapeutic response within the tumour microenviron- orthotopic mouse models of breast, thymus and lung ment through the suppression of an adaptive wound cancer, and melanoma14. healing response, but could also substantially reduce Thus, cathepsin activity is crucial both for the induc- dose-limiting toxicity. tion of a reactive and supportive tumour stroma in the context of therapeutic treatment and as a potent effec- Therapeutic targeting of cathepsins in cancer tor in this co‑opted wound repair response. Moreover, In the steady state of a normal healthy organism, defi- one of the major toxicities associated with chemothera- ciency of a specific cathepsin seems to be buffered by peutic agents — that is, intestinal mucositis — exhib- enzymatic redundancy and/or biological compensation its a similar pattern of treatment-induced increased as all individual cysteine cathepsin knockout mice that cathepsin activity. The pan-cathepsin inhibitor E-64 have been generated to date are viable62,64,121–124. In the

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context of various disease pathologies, however, it is evi- microenvironment20,24,25, and inhibition of these simul- dent that dysregulated activity of individual cathepsins taneously should in principle decrease the likelihood of can have considerable consequences. This important the development of acquired resistance. distinction makes cathepsins attractive pharmacological By contrast, treatment of the KPC mouse model of targets for the treatment of cancer, given that the poten- pancreatic ductal adenocarcinoma with E-64 as a mono- tial for serious side effects is low and consequently there therapy had no effect. Use of the MMTV-PyMT trans- should be a substantial therapeutic index. Moreover, as genic mouse model of breast cancer demonstrated that cathepsins appear to have pleiotropic roles in the promo- JPM-OEt had no efficacy alone28,134; however, this seems tion of tumour progression in certain cancers, they are to be the result of some limitations with regard to drug truly crucial effectors in the tumour microenvironment delivery and effective targeting. In support of this, use of that allow the inhibition of multiple tumorigenic pro- a ferri-liposome system of drug delivery demonstrated cesses through a single therapeutic target. As an exam- improved efficacy for JPM-OEt monotherapy in this ple, the apical role of CTSK in remodelling the bone preclinical model135. In vitro evidence has also demon- tumour microenvironment has led to the development strated efficacy of the peptide-based cathepsin inhibi- of a CTSK-specific small molecule inhibitor, odanacatib, tor Z‑Phe-Gly-NHO‑Bz as a cancer cell death-inducing for the treatment of breast-to‑bone metastasis125. agent in leukaemia and lymphoma as well as in neuro- This pharmacological development strategy, which blastoma cell lines136,137. Ultimately, however, our current focuses on highly specific, single-target inhibitors, is understanding of which cancers might respond clini- representative of the current approach to the targeting cally to cathepsin inhibitors, either selective or broad- of proteases as cancer therapeutics. The clinical failure of spectrum, is based largely upon empirical data and broad-spectrum MMP inhibitors126, due in part to inhibi- therefore requires further investigation. tion of both tumour-promoting and tumour-suppressive MMPs127, has led to caution in the development of thera- Conclusions and perspectives peutic strategies targeted against proteases in general, Increased activity of cathepsin proteases is a common particularly the use of broad-spectrum inhibitors. This phenotype associated with many different types of can- concern should also be noted for cathepsins, given the cer. This increased activity includes increases in both finding that CTSL has either tumour-promoting or intralysosomal and extralysosomal cathepsin activity, tumour-suppressive functions in different preclinical alternative cathepsin trafficking and the recruitment models24,70,72 (TABLE 2). Currently several pharmaceutical of infiltrating immune cells that express and secrete companies are developing selective or specific inhibitors cathepsins at high levels. Although the effects of this that target individual cathepsins for clinical use in cancer dysregulated enzymatic activity appear to be largely as well as in other indications including bone disorders, tumour promoting, there are clear instances of cath- Alzheimer’s disease, inflammation-associated pain and epsins exhibiting tumour suppressive activity. Indeed autoimmune diseases128–130. as our understanding of the protease web increases, In preclinical models, however, the use of pan- and our identification of cathepsin substrates becomes cysteine cathepsin inhibitors that target multiple family more comprehensive, it is likely that we will find that members has defined certain types of cancer for which many of these phenotypes are in fact composites of cathepsin inhibitors may have efficacy as a monotherapy various tumour-promoting and tumour-suppressing as opposed to those for which these agents will probably activities. Moreover, an improved understanding of the prove ineffective on their own. For example, treatment of integration and functionality of cathepsin proteases RIP1‑Tag2 mice with the pan-cathepsin inhibitor JPM- within the protease web will be crucial to understand- OEt significantly reduced tumour burden, angiogenesis ing their specific role in proteolysis globally. Matters and invasion23. Additionally, E-64, the natural product are further complicated by the fact that it remains analogue of JPM-OEt, which is also a pan-cathepsin unclear whether the connectivity and topology of the inhibitor, prevented colonization of the liver by Lewis protease web is meaningfully altered within the tumour lung carcinoma cells in an experimental metastasis microenvironment. model131. In comparison with broad-spectrum MMP Our understanding of the role of crucial cathepsin inhibitors, which were also evaluated in the RIP1‑Tag2 substrates is similarly incomplete. In vitro cleavage assays model132, pan-cathepsin inhibitors were substantially are of limited use in the identification of biologically more effective in impairing all the tumorigenic pro- relevant cathepsin substrates, as they cannot address cesses assessed23. Moreover, MMP ablation was shown the likelihood of the proteolytic reaction occurring in to lead to compensatory increases in cathepsins, in the relevant tissue microenvironment. At the most basic association with markedly more invasive PanNETs133. level, differential substrate expression and localization Broad-spectrum cathepsin inhibitors therefore have a will dictate the probability of cleavage by cathepsins in a notable advantage in the context of blockade of inva- given tissue. To this end, new proteomics resources such sion as illustrated by this example, and by the evidence as the Human Protein Atlas138 will hopefully shed light discussed above of intra-family compensation when on the tissue specificity of cathepsins and their target individual cathepsins are selectively ablated. Thus, cath- substrates. Additionally, experimental techniques that epsins represent a crucial target for collective blockade attempt to simultaneously measure protease activation, in this particular context, given that they direct multi- levels of endogenous inhibitors and substrates, and their ple mechanisms of cancer cell invasion in the PanNET cleavage in vivo have been developed and represent a

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significant advance in the study of protease biology139. improved chemotherapeutic benefit. An alternative strat- The field of cathepsin protease research is thus ripe for egy has also been proposed by which cathepsin activity the application of complex analysis to large compre- and the lysosome can be exploited as a weakness for hensive datasets to generate a more integrated view of the tumour82. Lysosomal destabilizing agents have the cathepsin activity within the tumour microenvironment. potential to form a new class of anticancer agents with From a translational perspective, the integration of good selectivity over normal tissue. Similarly, prodrugs cathepsin-targeting strategies into established chemo- have been designed to exploit the high cathepsin pro- therapeutic regimens seems to hold substantial promise. teolytic activity within the tumour and to restrict drug The dual nature of cathepsins as cell death effector mol- activation to these regions140–142. ecules and survival factors, however, indicates that this Fundamentally, the dysregulation of cathepsins is strategy should be approached with caution. Although a crucial component of neoplastic transformation and there is considerable potential for a decrease in the tumorigenesis. It is the inherent nature of proteases to variability of response and the suppression of adaptive degrade or irreversibly alter their target substrates. In resistance by the inhibition of cathepsins, it is important this manner, cathepsins exhibit both of these properties to determine the consequences of such an approach. It by recycling proteins for cancer cell survival and pro- will be crucial to fully characterize which chemother- liferation, as well as irreversibly modifying the tumour apeutic agents are sensitive to modulation by cath- microenvironment, which leads to growth, invasion epsins as opposed to those that depend on cathepsins and disease progression. Cathepsins act as a ‘biological to mediate their cytotoxic functions. Furthermore, as ratchet’ that progressively drives the microenvironment the role of the innate and adaptive immune systems to malignancy. Our understanding of the specific roles in therapeutic response becomes increasingly appreci- of individual cathepsins in different cancer types, micro- ated we must consider how cathepsin inhibition might environments and stages in malignant progression has affect this process. Do cathepsins contribute to a more expanded substantially in the past decade. In the next immunogenic or an immunologically silent cancer cell decade we hope to achieve an integrated understanding death? Will cathepsin inhibitors alter antigen presen- of cathepsin activity in the tumour microenvironment tation in a manner that suppresses the immunological and to determine the specific instances for which the response? These are questions for which answers must reliance of cancers upon cathepsins represents a fatal be found in order to effectively target these proteases for weakness that can be therapeutically exploited.

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