Chapter 15 Roles of Cysteine in Tumor Progression: Analysis of Cysteine Knockout Mice in Cancer Models

Thomas Reinheckel*, Vasilena Gocheva*, Christoph Peters, and Johanna A. Joyce

Abstract Cysteine are a family of proteases that are frequently upregu- lated in various human cancers, including breast, prostate, lung, and brain. Indeed, elevated expression and/or activity of certain cysteine cathepsins correlates with increased malignancy and poor patient prognosis. In normal cells, cysteine cathe- psins are typically localized in lysosomes and other intracellular compartments, and are involved in degradation and processing. However, in certain diseases such as cancer, cysteine cathepsins are translocated from their intracellular com- partments to the cell surface and can be secreted into the extracellular milieu. Pharmacological studies and in vitro experiments have suggested general roles for the cysteine cathepsin family in distinct tumorigenic processes such as angio- genesis, proliferation, apoptosis, and invasion. Understanding which individual cathepsins are the key mediators, what their substrates are, and how they may be promoting these complex roles in cancer are important questions to address. Here, we discuss recent results that begin to answer some of these questions, illustrating in particular the lessons learned from studying several mouse models of multistage carcinogenesis, which have identified distinct, tissue-specific roles for individual cysteine cathepsins in tumor progression.

T. Reinheckel, C. Peters Institut fu¨r Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universita¨t, D-79104 Freiburg, Germany V. Gocheva, J.A. Joyce Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA * These authors made an equal contribution

D. Edwards et al. (eds.), The Cancer Degradome. 279 # Springer Science þ Business Media, LLC 2008 280 T. Reinheckel et al.

Introduction

Proteolytic degradation is critically important during tumor development to facili- tate angiogenesis, invasion, and metastasis, biological processes known to require localized matrix remodeling. A broader role for in other aspects of tumor development, including promotion of cell proliferation and resistance to apoptosis, is increasingly recognized, emphasizing the importance of identifying the involved and understanding their mechanism of action. Aspartic, cysteine, serine, threonine, and matrix metalloprotease (MMP) families have been evaluated in multiple cancer types, with the major focus having been on the MMPs. Recently, however, cathepsins of the cysteine family have received increased atten- tion for their proposed roles in angiogenesis, apoptosis, cell proliferation, and tumor invasion (Mohamed and Sloane 2006, Gocheva and Joyce 2007). In this chapter, we discuss recent analyses of the effects of cysteine cathepsin deletion in several mouse models of multistage carcinogenesis, which have uncovered distinct, tissue-specific roles for individual cysteine cathepsins in tumorigenesis. Cysteine cathepsins are a group of enzymes that belong to the family of cysteine proteases. There are 11 members in humans and 18 in mice (Table 15.1), all of which share a conserved catalytic site formed by cysteine, histidine, and asparagine residues (Lecaille et al. 2002). Despite similarities in sequence and fold, cysteine cathepsin family members differ among each other in specificity. While most of them are endopeptidases, some possess exopeptidase activity. For example, cathepsin X is a carboxypeptidase, while can cleave dipeptides from the N-terminus of its protein substrates. Cathepsins B and H, however, have both endo- and exopeptidase activity. This difference in specificity among family mem- bers could influence their individual ability to cleave certain substrates, while ensuring efficient and complete degradation of by their collective action in the lysosomes. Therefore, it was initially believed that the major function of cysteine cathepsins is nonspecific, terminal protein proteolysis. However, we now know from the studies of cathepsin knockout mice that these enzymes play impor- tant, nonredundant roles in many other physiological and pathological processes (discussed in detail in the next section). Cysteine cathepsins are synthesized as inactive precursors containing an amino- terminal signal peptide that mediates their transport across the endoplasmic reticu- lum membrane (Turk et al. 2000). All cysteine cathepsins possess N-glycosylation sites, which are used to target the enzymes to the lysosomal compartment via the mannose 6-phosphate receptor pathway. Activation is fully achieved once the enzymes reach the acidic environment of the late endosomes or lysosomes, where the propeptide that blocks access of substrates to the catalytic cleft of the proen- zyme is removed (Coulombe et al. 1996, Cygler et al. 1996, Sivaraman et al. 1999). Endoproteolytic removal of the propeptide is accomplished by either autocatalytic processing (in the case of the endopeptidases) or limited proteolysis by other proteases. This process is triggered by low pH and is enhanced by the presence of glycosaminoglycans (GAGs) (Wiederanders and Kirschke 1989). GAGs support 15 Roles of Cysteine Proteases in Tumor Progression 281

Table 15.1 Nomenclature and expression patterns of cysteine cathepsins in human and mouse Protease (alternative names) Human Mouse Expression pattern (11) (18) CTSB CtsB Ubiquitous Cathepsin C (J, Dipeptidyl CTSC CtsC Ubiquitous peptidase I) CTSF CtsF Ubiquitous CTSH CtsH Ubiquitous (O, O2) CTSK CtsK Osteoclasts, lung-epithelium, thyroid gland CTSL – Ubiquitous (V) CTSL2 CtsL Thymus, testis, cornea, epidermis, macrophages CTSO CtsO Ubiquitous CTSS CtsS Lymphatic tissues, antigen-presenting cells (APC), muscle (Lymphopain) CTSW CtsW Natural killer (NK) cells, cytotoxic T lymphocytes Cathepsin X (Z, P, Y) CTSZ CtsZ Ubiquitous Cathepsin J – CtsJ Placenta Cathepsin M – CtsM Placenta Cathepsin Q – CtsQ Placenta Cathepsin R – CtsR Placenta Cathepsin 1 – Cts1 Placenta Cathepsin 2 – Cts2 Placenta Cathepsin 3 – Cts3 Placenta Cathepsin 6 – Cts6 Placenta

autoactivation and activity of cysteine cathepsins at neutral pH, which is highly relevant for the activation of secreted procathepsins (Vasiljeva et al. 2005). Simi- larly, cathepsin K requires binding to chondroitin sulfate for optimal collagenolytic activity (Lecaille et al. 2002). Not all cysteine cathepsins are exclusively present in the lysosomes. For exam- ple, cathepsin W is retained in the endoplasmic reticulum (Ondr and Pham 2004), while an isoform of cathepsin L was found in the nucleus, where it can process the CDP/Cux transcription factor (Goulet et al. 2004). Moreover, in certain pathologi- cal conditions, cysteine cathepsins, which are considered to be intracellular pro- teases, have been detected at the cell surface (Mai et al. 2000) and cathepsin activity can also be detected in the extracellular milieu (Mort et al. 1985). In fact, early descriptions of cathepsin L referred to this protease as ‘‘major excreted protein (MEP)’’ of malignantly transformed fibroblasts (Mason et al. 1987). Cysteine cathepsin secretion from several cancer cell lines has also been reported, and will be reviewed in Chap. 29 (Lah et al.). Although the precise mechanism of cysteine cathepsin secretion is an area of active investigation, it has been shown that at the cell surface, cathepsins are associated with various binding partners such as integ- rins or annexin II in discrete regions in the plasma membrane such as caveolae 282 T. Reinheckel et al.

(Mai et al. 2000, Lechner et al. 2006). Procathepsin B can interact with the annexin II heterotetramer, a protein involved in plasminogen activation. The varied locali- zation of cysteine cathepsins in cancer cells will undoubtedly influence the range of substrates available, and therefore the mechanisms by which they contribute to a wide range of biological processes, including key steps in the progression of primary and metastatic tumors. In addition to differences in their subcellular localization, cysteine cathepsin family members also exhibit a diverse tissue distribution. The majority of cathe- psins are ubiquitously expressed in most normal tissues, suggesting housekeeping functions (Lecaille et al. 2002) (Table 15.1). In contrast, the expression of other members, in particular cathepsin K, S, L2, and W, is restricted to specific cell or tissue types (Table 15.1) where they carry out specialized roles. Cathepsin K is primarily localized to osteoclasts (Drake et al. 1996), while cathepsin S is present mostly in lymphatic tissues and immune cell types such as antigen-presenting cells (Shi et al. 1994). Cathepsin L2, which is the orthologue of mouse cathepsin L, is specifically expressed in the thymus, testis, and corneal epithelium (Adachi et al. 1998; Santamaria et al. 1998; Yasuda et al. 2004; Cheng et al. 2006). Cathepsin W also shows a restricted expression pattern in cytotoxic lymphocytes and natural killer cells (Brown et al. 1998). Finally, there are eight cysteine cathepsins unique to the mouse with placental-specific expression (Deussing et al. 2002). Cell-type- specific cysteine cathepsin expression likely contributes to some unique cathepsin functions, which are discussed in the next section. Given the destructive potential of the cysteine cathepsins, several safeguarding mechanisms exist to protect normal cells from uncontrolled proteolysis: they are synthesized as inactive precursors, compartmentalized within lysosomes, and are targets of endogenous inhibitors, which can bind any inappropriately activated cathepsins. Several families of endogenous inhibitors exist including stefins, cysta- tins, and kininogens (Turk et al. 2001). Stefins (A and B) act mostly intracellularly, while the kininogens and cystatins (C, D, E/M, F, S, SN, and SA) are extracellular proteins. Cystatin C is the most potent inhibitor of cathepsins B, H, L, and S, and displays broad tissue distribution (Kopitar-Jerala 2006). Other inhibitors include thyropins and the general inhibitor a2-macroglobulin (Turk et al. 2000). Maintain- ing the appropriate balance between cysteine cathepsins and their inhibitors is essential given the important roles these proteases can play in numerous pathological conditions when misregulated, particularly in cancer development.

Phenotypes of Cysteine Cathepsin Knockout Mice

In recent years, knockout mice have been generated for nearly every member of the cysteine cathepsin family (Saftig et al. 1998, Pham and Ley 1999, Halangk et al. 2000, Roth et al. 2000, Ondr and Pham 2004, Tang et al. 2006, Reinheckel et al. unpublished data). Phenotypic analysis of these mice has revealed specific and nonredundant roles for individual cysteine cathepsins. While protein degradation in 15 Roles of Cysteine Proteases in Tumor Progression 283 the lysosomes is not the exclusive property of any one cathepsin, as there is no widespread defect in that process in any of the cysteine cathepsin null animals, other physiological functions such as epidermal homeostasis, normal cardiac and brain function, antigen presentation, and apoptosis are affected. For example, cathep- sin S null (CtsS/) mice have defects in MHC class II-associated antigen proces- sing and presentation, indicating an important role for this protease in mediating humoral immunity (Shi et al. 1999). Cathepsin L was shown to be involved in skin morphogenesis, hair follicle morphogenesis and cycling, and heart function since its ablation causes epidermal hyperplasia and periodic hair loss (Roth et al. 2000), and CtsL/ mice develop cardiomyopathy with age (Stypmann et al. 2002; Petermann et al. 2006). Cathepsin C is a processing involved in the activation of several serine proteases such as granzymes A and B, and CtsC/ mice have corresponding defects in cytotoxic T cell-induced apoptosis (Pham and Ley 1999). Cathepsin K null (CtsK/) mice have impaired resorption of the bone matrix and develop osteopetrosis, which fits well with the restricted expression of cathepsin K in osteoclasts (Saftig et al. 1998). The generation of cathepsin F null (CtsF/) mice was recently reported (Tang et al. 2006) and proposed as a model of late-onset neurological disease. CtsF/ mice develop progressive defects in motor coordination and hind leg weakness, and the authors infer this phenotype may result from the accumulation of lipofuscin in CNS neurons (Tang et al. 2006). On the contrary, mice mutant for other family members such as cathepsins B, W, H, and X appear normal (Ondr and Pham 2004, Reinheckel et al. unpublished data). However, further detailed analyses may be required to identify phenotypic con- sequences of their ablation, if indeed there are any. As an example, the phenotype of cathepsin B null (CtsB/) mice is very subtle and these animals can be distin- guished from their littermates only when subjected to pathological stress, such as experimental pancreatitis and liver injury, to which they are resistant (Halangk et al. 2000, Guicciardi et al. 2001).

Cysteine Cathepsins in Cancer

Cysteine cathepsin upregulation has been reported in many human tumors, includ- ing breast (Poole et al. 1978, Castiglioni et al. 1994, Bervar et al. 2003), lung (Werle et al. 1999; Fujise et al. 2000), brain (Rempel et al. 1994, Strojnik et al. 1999), gastrointestinal (Watanabe et al. 1989, Liu et al. 1998, Ebert et al. 2005), prostate (Sinha et al. 1995, Nagler et al. 2004) cancers and melanoma (Kos et al. 1997, Frohlich et al. 2001). The increased expression of certain cysteine cathepsins, such as cathepsin B and cathepsin L, has frequently been positively correlated with a poor prognosis for patients with a variety of malignancies (Saad et al. 1998, Frohlich et al. 2001, Harbeck et al. 2001), although contrary results have also been reported (Nasu et al. 2001). Moreover, increased antigen levels of cysteine cathepsins in the serum have been reported for several types of cancer (Thomssen et al. 1995, Lah et al. 2000, Schweiger et al. 2000, Miyake et al. 2004), indicating 284 T. Reinheckel et al. their potential use as prognostic biomarkers. A detailed discussion of cysteine cathepsins in human cancer can be found in Chap. 29 (Lah et al.). However, while clinical association studies emphasize the importance of under- standing the roles of these proteases in cancer, they do not provide causal insights into the in vivo functions of cysteine cathepsins in tumor initiation and progression. Thus, we set out to investigate the roles of a subset of cathepsins that had been implicated in key tumorigenic processes (Joyce et al. 2004), by breeding cathepsin knockout mice with mouse models of human pancreatic islet cell, breast, or skin carcinogenesis (Fig. 15.1). Tumor progression and metastasis in the cathepsin deficient, cancer-prone mice were subsequently analyzed and will be discussed in detail below. The hallmarks of malignancy in the three mouse models of human cancer are as follows:

RIP1-Tag2 Model of Pancreatic Islet Cell Carcinogenesis

Carcinomas of the endocrine islets in the pancreas are induced in transgenic mice by expression of the simian virus type-40 large and small T-antigens (Tag) under the control of the rat insulin promoter (RIP) (Hanahan 1985). In the RIP1-Tag2 (RT2) mice, approximately 50% of the 400 pancreatic islets of newborn mice develop hyperplasia during the first month of life. Subsequently, 45–50 of these islets induce neovascularization, in a process that is termed ‘‘angiogenic switching’’ (Hanahan and Folkman 1996), in which the preneoplastic lesions develop an independent blood supply, a step that is essential for subsequent tumor develop- ment. Approximately 10 islet cell tumors arise per mouse by 12–14 weeks of age, which can be classified as encapsulated, microinvasive, and invasive carcinomas that are, however, rarely metastatic (Lopez and Hanahan 2002) (Fig. 15.1).

MMTV-PyMT Model of Mammary Epithelial Carcinogenesis

Breast cancer in this model is induced by transgenic expression of the Polyoma virus middle-T-oncogene (PyMT) in the ductal epithelium of the mammary gland (Guy et al. 1992). The specificity of oncogene expression is determined by the long terminal repeat (LTR) promoter of the mouse mammary tumor virus (MMTV). This viral promoter is costimulated by ovarian steroid hormones and therefore tumor- igenesis is initiated in female mice during puberty at 4–5 weeks of age. The first pathological alterations, atypical hyperplasias and adenosis, are still benign. These lesions progress within a few weeks to premalignant ductal carcinoma in situ (DCIS) and at later stages to malignant invasive ductal carcinomas (IDC) (Fig. 15.1). At 14 weeks of age, all female MMTV‐PyMT mice exhibit large and mostly poorly differentiated IDCs in each of their ten mammary glands. At this stage, all mice have developed multiple metastases in the lungs. Metastasis also occurs in the axillary and cervical lymph nodes; however, the penetrance of lymph node metastases is not 100%, as for lung metastases (Almholt et al. 2005). 5Rlso ytiePoessi uo Progression Tumor in Proteases Cysteine of Roles 15 285

Fig. 15.1 Transgenic mouse models of cancer used to study the effects of cathepsin ablation on tumorigenesis 286 T. Reinheckel et al.

K14-HPV16 Model of Human Skin and Cervical Cancer

Human papillomaviruses (HPV) cause proliferative lesions in the skin and squa- mous mucosa, which are mostly hyperplasias, papillomas, and condylomas. How- ever, epithelial lesions of some HPV subtypes, with HPV16 among them, often progress to high-grade dysplasias and occasionally to carcinomas (zur Hausen 1987). Transgenic mice expressing the early region of the HPV16 genome (includ- ing the critical oncogenes E6 and E7) in basal epithelial cells under control of the human cytokeratin 14 promoter (K14) undergo the classical sequence of malignant progression during the first 12 months of life (Fig. 15.1). Female as well as male K14-HPV16 mice develop hyperplasia and papillomas in the skin (Arbeit et al. 1994). In the FVB/n genetic background, subsequent development of dysplasias with abundant neoangiogenesis results in 50% of all mice developing squamous cell carcinomas that metastasize to regional lymph nodes at a frequency of about 20% (Coussens et al. 1996). In addition to the epidermal squamous cell carcinomas, cervical cancers can also be induced in these mice by costimulation with estrogen. Notably, most of the tumor-bearing mice develop only one squamous cell carcino- ma despite the fact that the HPV16 oncogenes are expressed throughout the epidermis of the entire animal. Thus, this mouse model is of considerable value for investigation of additional factors that alter the incidence, progression, and metastasis of HPV16-induced tumors. Despite the differences between the three cancer models in terms of oncogenes, tissue specificities, and timescales of cancer development, they all undergo a sequential, multistep evolution from normal (although oncogene-positive) tissues to invasive cancer. This is a highly important conceptual distinction from the frequently used models involving orthotopic or ectopic transplantation of ex vivo maintained and selected cancer cells. Xenograft studies in particular, which involve injection or transplantation of human cancer cell lines into immunodeficient mice, are unlikely to recapitulate essential interactions between the developing tumor and its surrounding tissue microenvironment. Moreover, the primary tumor models studied by us exhibit the complete and natural actions of the innate and adaptive immune system in response to the growing tumor. Aspects of this immune response can be proangiogenic and protumorigenic, while other immune cells are tumorici- dal (de Visser et al. 2006). Since cysteine cathepsins are highly expressed in immune cells (Table 15.1) and have critical roles in MHCII-mediated antigen presentation, it is essential to investigate these proteases in immunosufficient models, such as in the experimental animals covered in this chapter.

Analysis of Cysteine Cathepsin Deficiency in Cancer-Prone Mice

In order to determine the involvement of cysteine cathepsins in cancer, several labs have crossed cathepsin knockout mice to different models of tumorigenesis, which have revealed distinct roles for these proteases in regulating cancer pathogenesis 15 Roles of Cysteine Proteases in Tumor Progression 287 and invasion. In this section, we will describe the phenotypic analysis for each of these crosses, and in the following section, we will discuss the mechanistic insights into cysteine cathepsin functions in vivo that have been revealed by these genetic experiments.

Cathepsin Deficiencies in the RIP1-Tag2 Pancreatic Cancer Model

The most thorough genetic analysis of the involvement of cathepsins in tumorigen- esis to date has been completed in the RIP1-Tag2 (RT2) model. Increased expres- sion of six members of the cysteine cathepsin family (cathepsins B, C, H, L, S, and X) was reported in this model over the course of tumor progression (Joyce et al. 2004). The collective importance of the cathepsin family was demonstrated using a broad-spectrum cysteine cathepsin inhibitor, which significantly perturbed all stages of tumor development. Subsequently, mice null for cathepsins B, C, L, S (Gocheva et al. 2006), H, and X (Gocheva and Joyce unpublished data) were crossed with RT2 mice in order to determine the individual functions, if any, of these cysteine cathepsins in pancreatic islet cell cancer. Indeed, it was found that RT2 mice null for cathepsin B, L,orS had a significant reduction in tumor burden and general perturbations throughout the course of tumor development (Table 15.2). This detailed analysis also revealed some process-specific functions of individual cathepsin family members. Specifically, we showed that deletion of cathepsin B or S reduced angiogenic switching and caused defects in the tumor vasculature, including decreased micro- vessel density (MVD), indicating that these proteases are not only important for neovascularization but are also essential for the continuous maintenance of tumor angiogenesis. An independent study reported a similar decrease in tumor burden in CtsS / RT2 mice and also suggested a functional role for this enzyme in angio- genesis (Wang et al. 2006). Moreover, ablation of cathepsins B, L,orS caused a pronounced increase in tumor cell apoptosis, which correlated well with the significantly decreased tumor burden in these mice (Gocheva et al. 2006) (Table 15.2). Another process that was affected was tumor cell proliferation, which was significantly decreased in CtsB/ and CtsL/ RT2 mice, again closely correlating with the substantial reduction in their tumor burdens (Table 15.2). Surprisingly, proliferation was not affected in the CtsS/ RT2 animals, indicating that the nearly 50% decrease in tumor burden is due to a combination of increased cell death and defects in angiogenesis (Gocheva et al. 2006). Critically, deletion of any one of these three cathepsins (B, L, or S) caused a significant reduction in the invasiveness of the resulting tumors, with a marked decrease in the percentage of microinvasive as well as frankly invasive lesions. Thus, in these three cathepsin knockouts, not only was there a significant reduction in pancreatic tumor burden, but also the tumors that did arise were mostly benign, encapsulated lesions (Gocheva et al. 2006). These results demonstrated that 288

Table 15.2 Summary of the effects of cathepsin ablation in the different mouse models of human cancer analyzed CtsB / CtsL / CtsS / CtsC / RIP1-Tag2a MMTV-PyMTb RIP1-Tag2a K14-HPV16c RIP1-Tag2a,d RIP1-Tag2a Development of premalignant lesions Reduced Reduced Reduced Enhanced Reduced No change Tumor volume Reduced Reduced Reduced Accelerated development Reduced No change Metastatic tumor burden N/Ae Reduced N/Ae Enhanced N/Ae N/Ae Proliferation Reduced Reduced Reduced Enhanced No change No change Apoptosis Enhanced No change Enhanced No change Enhanced No change MVDf/angiogenic switch Reduced No change No change No change Reduced No change Tumor grade/invasion Reduced Reduced Reduced Increased Reduced Reduced The data summarized in this table is compiled from the following references: a Gocheva et al. (2006) b Vasiljeva et al. (2006) c Lohmuller and Reinheckel, manuscript in preparation d Wang et al. (2006) e Tumors in the RIP1-Tag2 model very rarely metastasize f Microvessel density (MVD) .Rihce tal. et Reinheckel T. 15 Roles of Cysteine Proteases in Tumor Progression 289 mutating any one of the cysteine cathepsin proteases B, L, or S interferes with the progression of benign lesions to invasive carcinomas, indicating that each enzyme plays an important and nonredundant role in the process of tumor invasion. Interestingly, although cathepsin C was identified as one of the six cysteine cathepsins upregulated during islet tumor progression (Joyce et al. 2004), when RT2 mice are null for cathepsin C, there was no effect on any of the parameters analyzed (Gocheva et al. 2006) (Table 15.2). This result illustrates the importance of thorough functional validation for candidates arising from microarray profiling of cancers, as there will naturally be whose expression is altered as a result of changes in the transcriptional program, but which do not functionally contribute to cancer development.

Cathepsin B Deficiency in the MMTV-PyMT Mammary Cancer Model

The involvement of cathepsin B in promoting tumorigenesis has also been estab- lished in the Polyoma middle-T-oncogene (PyMT) mouse model of mammary cancer, indicating that cathepsin B plays important protumorigenic roles in cancers of different tissue types and different cells of origin. In this model, it was deter- mined that deleting one or both alleles of CtsB delayed the onset of mammary tumor formation and caused a reduction in tumor growth rates, with the resulting lesions being smaller in size (Vasiljeva et al. 2006). This impaired tumor progres- sion is caused by reduced proliferation rates and diminished invasive potential of CtsB/ cancer cells, while apoptotic rates did not differ between the CtsBþ/ þ and CtsB / tumors (Table 15.2). An important characteristic of the PyMT model is that the tumors spontaneously metastasize to the lung, thus allowing the authors to investigate whether cathepsin B plays a role in metastasis, in addition to its involvement in primary tumor growth. In fact, ablation of one or both copies of CtsB caused a reduction in the volume of pulmonary metastases to 45% and to 65%, respectively. The authors extended their analysis of the role of cathepsin B in the process of tumor cell seeding by using a lung colonization experimental assay, where LacZ-tagged primary PyMT cells þ were intravenously injected into wild-type female mice. Injection of CtsB / PyMT or CtsB/ PyMT cells resulted in a significant decrease in both the number and the size of the lung colonies compared to wild-type PyMT cells, indicating the importance of cathepsin B in the formation and growth of distant metastases (Vasiljeva et al. 2006).

Cathepsin L Deficiency in the K14-HPV16 Skin Cancer Model

While the aforementioned studies demonstrated protumorigenic functions for a subset of cysteine cathepsins, with their deletion causing significant defects in tumor development, this seems to depend on the particular type of cancer and 290 T. Reinheckel et al. mouse model investigated. A recent study assessed the functional contribution of cathepsin L to epidermal carcinogenesis by crossing CtsL/ mice with transgenic K14-HPV16 mice (Lohmuller and Reinheckel manuscript in preparation). Surpris- ingly, ablation of CtsL actually enhanced tumor progression, with an earlier onset of formation of palpable skin tumors (Table 15.2). Furthermore, these epithelial tumors were more malignant and there was increased metastasis to the axillary lymph nodes compared to wild-type K14-HPV16 mice. While angiogenesis and inflammation during neoplastic progression were unaffected, there was a significant increase in proliferation of the epidermis in CtsL/ K14-HPV16 mice compared to CtsLþ/þ K14-HPV16 controls. Although these findings may seem surprising as they challenge the large body of evidence supporting procancerous roles for cysteine cathepsins, including cathepsin L, they may actually be related to the fact that cathepsin L plays a major role in the maintenance of epidermal homeostasis (Roth et al. 2000). In fact, CtsL/ mice without any oncogene expression also develop epidermal hyperproliferation that can be normalized by keratinocyte-specific reex- pression of the protease, indicating a cell-type-specific role of cathepsin L in the regulation of epidermal proliferation (Reinheckel et al. 2005). Therefore, detailed and comprehensive analyses are necessary in order to fully understand the roles that individual cysteine cathepsin family members, such as cathepsin L, play in multiple types of cancer, as the involvement of certain cathe- psins may vary between the different models analyzed. For example, it could be very interesting to examine the effects of cathepsin L deletion on cervical carcinogenesis in the K14-HPV16 mice and ask whether the tumor-suppressing effects of this cysteine cathepsin are specific to the skin, or are observed in other epithelial tissues. Similarly, are there unique protective functions for cysteine cathepsins other than cathepsin L in preventing transformation of the skin epithelium? Analysis of K14- HPV16 mice mutant in other cathepsin family members will allow us to determine whether there are examples where these proteases are tumor-suppressive, or whether this is a unique function of cathepsin L. Certainly the continued investigation of cysteine cathepsin function in these and other transgenic and ‘‘knock-in’’ models of cancer is essential to address the general importance of cysteine cathepsins in cancer promotion, a critical question that should be answered in preclinical models before advancing to the clinic with cysteine cathepsin inhibitors.

Mechanistic Insights into Cysteine Cathepsin Function from Knockout Mice

The studies described above in the cathepsin-deficient mice have uncovered some process- and tissue-specific roles for cysteine cathepsin family members in a variety of mouse models of cancer to date, and the subsequent investigation of the biological mechanisms underlying these processes in vitro has provided novel insights into cysteine cathepsin functions in tumorigenesis. In some cases, the 15 Roles of Cysteine Proteases in Tumor Progression 291 investigation of cathepsin deficiencies in vivo has confirmed candidates suggested from the previous biochemical analysis of cathepsin substrates, and in other cases the knockout mice have enabled the identification of completely new substrates, as will be discussed below. Angiogenesis and invasion are two of the stages in tumor development that have long been known to require proteoloytic activity. As dis- cussed in the previous section, analysis of several cathepsin knockouts in different tumor models has confirmed important roles for cysteine cathepsin proteases in each of these processes.

Angiogenesis

The process of new blood vessel formation, termed angiogenesis or neovascular- ization, is critical for the growth of a tumor beyond 1–2 mm3 in size (Hanahan and Folkman 1996, Carmeliet and Jain 2000). Angiogenesis is typically initiated by the release of proangiogenic growth factors, such as vascular endothelial growth factor (VEGF) from the tumor. Endothelial cells migrate into the growing lesion and divide and differentiate to form new blood vessels, which may be additionally supported by pericytes and smooth muscle cells. Induction of proteolysis is neces- sary for the controlled degradation of the extracellular matrix (ECM) and vascular basement membrane (BM), a critical step in vessel sprouting. The outcome of whether angiogenesis is activated ultimately depends on the balance between proangiogenic (VEGF, FGFs, proteases, etc.) and antiangiogenic (endostatin, tum- statin, thrombospondin, etc.) factors. To date, cathepsin B and S have been shown to be involved in tumor angiogenesis in the RT2 model (Table 15.2). One mechanism by which cathepsin B and S could promote blood vessel formation is through proteolysis of BM constituent proteins, such as laminin, collagen IV, and fibronectin, which cathepsin B has been shown to cleave in vitro (Buck et al. 1992). Moreover, CtsS / endothelial cells show a decreased ability to cleave elastin and collagen IV, leading to an impaired ability of these cells to invade through Matrigel or collagen I gel membranes (Shi et al. 2003). Therefore, CtsS/ mice have defects in microvessel formation under different conditions, including wound healing and tumor angiogenesis. A more direct mechanism by which cathepsins might influence the formation of new blood vessels is by inactivating angiogenic inhibitors or by processing proan- giogenic factors. For example, protein levels of three antiangiogenic factors, tum- statin, arresten, and canstatin, are increased in the CtsS/ RT2 tumors compared to þ þ the CtsS / RT2 controls (Wang et al. 2006). Biochemical data confirmed that cathepsin S degrades arresten and canstatin in vitro, in addition to generating proangiogenic g2 fragments from laminin-5, a common basement membrane com- ponent (Wang et al. 2006). Similarly another family member, cathepsin L, can generate endostatin, a well- known endogenous inhibitor of angiogenesis, from collagen XVIII in vitro (Felbor et al. 2000), thereby suggesting an active role for this protease in regulating 292 T. Reinheckel et al. angiogenesis. However, another report has suggested that after its rapid generation, cathepsin L can then degrade endostatin very efficiently in vitro (Ferreras et al. 2000). Cathepsin B was also found to cleave endostatin, though much less efficient- ly than cathepsin L, and might also participate in its degradation (Ferreras et al. 2000). Thus, the precise role of cathepsins in influencing the balance between the generation and destruction of this antiangiogenic factor, and others, in the complex tumor microenvironment still remains to be elucidated, and the analysis of the different cysteine cathepsin null tumor models discussed here now represents an ideal system to address this question. Cathepsin L was also shown to be required for neovascularization after ischemia by enhancing the ability of endothelial progenitor cells to invade ischemic tissues and incorporate into the newly forming vessels (Urbich et al. 2005). Whether cathepsin L plays a similar role in the neovascularization of tumors is still unknown, as the contribution of endothelial progenitor cells to tumor angiogenesis is still an area of active debate and may be very mouse model specific (see Nolan et al. 2007 for discussion). While we did not detect angiogenic defects in the few, small tumors that develop in the CtsL / RT2 mice (Gocheva et al. 2006) (Table 15.2), we know that islet cell tumors do not become dependent on new blood vessel formation until they require a significant expansion in size (Bell-McGuinn et al. 2007). Since the CtsL/ RT2 tumors were up to 90% smaller on average than the control RT2 tumors, it is possible that the critical switch in which angiogenesis is activated had not yet been triggered, resulting in unchanged microvessel density.

Tumor Invasion

Cysteine cathepsins may contribute not only to the invasion and migration of endothelial cells during angiogenesis, but they could also promote the dissemina- tion of cancer cells into the surrounding tissue. There have been several possible mechanisms proposed for how cathepsins could be involved in the process of invasion and metastasis including degradation of the ECM, activation of other proteases, or cleavage of cell–cell adhesion factors (Gocheva and Joyce 2007). Indeed, using the individual cathepsin null/RT2 tumors described above, we identified the cell-adhesion protein, E-cadherin, as a novel substrate for the proin- vasive cathepsins B, L, and S (Gocheva et al. 2006). E-cadherin is the principal component of adherens junctions that maintain cell–cell adhesion, thereby limiting cell migration (Cavallaro and Christofori 2004). In fact, downregulation of E- cadherin through mechanisms such as silencing, point mutations, or posttrans- lational modifications is a common hallmark of invasive cancers (Cavallaro and Christofori 2004). In RT2 tumors, E-cadherin protein expression is typically lost as lesions become progressively more invasive, particularly at the tumor margin (Perl et al. 1998). However, in RT2 tumors null for cathepsin B, L,orS, the levels of E- cadherin protein were maintained, in stark contrast to the control RT2 tumors or the CtsC / RT2 tumors (Gocheva et al. 2006). It was reasoned that posttranslational 15 Roles of Cysteine Proteases in Tumor Progression 293 cleavage of E-cadherin was perturbed in these cathepsin null RT2 mice, and subsequent biochemical analysis confirmed that E-cadherin is cleaved in its extra- cellular domain by cathepsins B, L, and S, but not cathepsin C. In other systems, cleavage of the extracellular portion of E-cadherin has been shown to abrogate its adhesive functions and promote tumor cell invasion (Cavallaro and Christofori 2004), and ongoing experiments should further clarify the exact mechanism of action for cathepsin-mediated E-cadherin cleavage and tumor cell invasion (Gocheva and Joyce unpublished data). Additional mechanisms by which cysteine cathepsins promote invasion may involve direct degradation of components of the ECM, thus creating space for the invading cells to migrate into. For example, cathepsins B and L were shown to cleave components of the BM/ECM including laminin (Lah et al. 1989), type-IV collagen (Buck et al. 1992, Guinec et al. 1993), fibronectin (Buck et al. 1992, Ishidoh and Kominami 1995), and tenascin-C (Mai et al. 2002), leading to limited proteolysis of the surrounding matrix. During cancer development, cysteine cathe- psins are often translocated to the cell surface where they can be associated with proteins such as annexin II, or secreted, as discussed in the Introduction. Once at the plasma membrane or the extracellular space, cysteine cathepsins have im- proved access to other cell-surface proteases or to their ECM substrates, although some studies have demonstrated that degradation of ECM components can also occur intracellularly (Sameni et al. 2000, 2001). Finally, cysteine cathepsins also participate in the activation of other proteases by initiating a proteolytic cascade leading to the cleavage of multiple downstream targets, which could collectively enhance the process of tumor invasion. In vitro experiments have shown that cathepsins cleave and activate MMP-1 and MMP-3 (Eeckhout and Vaes 1977), as well as convert the precursor form of urokinase-type plasminogen activator (uPA) to the active enzyme, which in turn catalyzes the cleavage of plasminogen into plasmin (Goretzki et al. 1992, Guo et al. 2002). Plasmin is a broad-spectrum serine protease that can also directly degrade ECM components and activate other MMPs (Rao 2003). Thus, the invasion of cancer cells is likely facilitated by a combination of these proteolytic mechanisms, including dissociation of cell surface adhesion complexes and degradation of the surrounding ECM to allow local invasion and ultimately tumor cell metastasis. The generation of tumor-bearing mice deficient in individual cysteine cathepsins represents the ideal experimental model to now determine the relative contributions of these different pathways in different tumor microenviron- ments under physiologically relevant conditions.

Tumor Cell Proliferation and Apoptosis

Tumor growth is largely influenced by the balance between tumor cell proli- feration and apoptosis, and previous in vitro data had suggested that certain cysteine cathepsins may have both proproliferative and antiapoptotic roles that 294 T. Reinheckel et al. could significantly contribute to increased tumor growth and malignancy. In fact, reduced tumor cell proliferation rates were found in CtsB/ RT2, CtsL/ RT2, and CtsB/ PyMT mice (Table 15.2), suggesting that these proteases might normally be activating growth factors or cleaving key proteins implicated in cell cycle progression. Indeed, cathepsin B was previously shown to proteolytically cleave and process the insulin-like growth factor-1 (IGF-1) in endosomes following IGF-1-induced IGF-1 receptor internalization, resulting in appropriate downstream cellular signaling (Authier et al. 2005). In addition, cathepsin L might also play a more direct role in proliferation through cleavage and activation of the transcription factor CDP/Cux-1, thus pro- moting cell cycle progression and cellular proliferation (Goulet et al. 2004). However, cathepsin L’s proproliferative role might be strictly tissue specific as CtsL/ mice exhibit epidermal hyperplasia (Roth et al. 2000) and CtsL/ HPV16 tumors have enhanced proliferation indexes (Reinheckel et al. unpublished data) as discussed above. In the skin, cathepsin L was shown to increase the recycling of growth factors in keratinocytes resulting in sustained keratinocyte proliferation due to the increased availability of growth factors (Reinheckel et al. 2005), again pointing toward the more specialized progrowth functions of this protease in the skin epithelium. The involvement of cathepsins in tumor cell proliferation has also been con- firmed indirectly by Wang and colleagues, who analyzed the phenotype of cystatin C null RT2 mice (Wang et al. 2006). Cystatin C inhibits cathepsins B, L, S, and H and its absence resulted in a general increase in cathepsin activity. It was found that cell proliferation was significantly increased in cystatin C / RT2 tumors, again indicating that at least some of the cathepsins repressed by this endogenous inhibi- tor have progrowth functions. In terms of the effects of cysteine cathepsins on apoptosis, it was shown that ablating cathepsins B, L,or S in the RT2 model resulted in a significant increase in programmed cell death (Gocheva et al. 2006) (Table 15.2). These results indicate that these enzymes could have antiapoptotic roles during tumor develop- ment, although it might be context and stimulus dependent since other studies have reported that under certain conditions cathepsins can actually promote cell death. In fact, cathepsin B acts as an executioner protease in TNF-alpha-induced tumor cell death in a fibrosarcoma cell line (Foghsgaard et al. 2001) and was able to trigger cytochrome c release from the mitochrondria in TNF-alpha-mediated hepatocyte apoptosis (Guicciardi et al. 2000), potentially explaining why CtsB / mice are resistant to TNF-alpha-induced liver injury (Guicciardi et al. 2000). However, additional cathepsin cleavage substrates need to be identified before the precise mechanism of action of these proteases in the process of cell death is fully understood, which could also help resolve whether the local cellular environment controls whether cysteine cathepsins are ultimately pro- or antiapoptotic. 15 Roles of Cysteine Proteases in Tumor Progression 295

Functional Compensation by Cathepsin Family Members in Cysteine Cathepsin Knockout Mice

One concern that is often raised in the generation and analysis of knockout mice for entire families, as for the cysteine cathepsins discussed here, is that functional compensation by other family members may occur to take over the role of the deleted gene, which could potentially obscure the mutant phenotype. Given this consideration, we have analyzed the total expression and activity of the cysteine cathepsin family in the different cathepsin null cancer-prone mice discussed above, to determine whether there is compensatory upregulation. Analysis of cysteine cathepsin expression and activity in the different cathepsin null RT2 tumors did not reveal any changes when compared to wild-type RT2 tumors (Gocheva et al. unpublished data). This was also the case in an independent analysis of CtsS/ RT2 tumor cell extracts by Wang et al. (2006). However, it was determined that ablation of cathepsin B in PyMT tumor cells results in increased levels of cathepsin X on the cell surface, suggesting that there might be some compensatory mechan- isms within the family (Vasiljeva et al. 2006). Surprisingly, there was no significant difference in cathepsin X mRNA or protein levels between wild-type PyMT and CtsB / PyMT animals, indicating that altered intracellular trafficking and redistri- bution of the enzyme is responsible for the increased levels on the cell membrane. These changes in cathepsin X levels seem to be functionally relevant since inhibi- tion of cathepsin X by a neutralizing antibody caused an even further reduction in invasion of CtsB/ PyMT cells in culture, indicating that there might be some redundancy in the action of certain proteases in tumorigenic processes. The generation of CtsB/; CtsL/ double-knockout mice has also indicated that these two cathepsins play redundant roles in the maintenance of the central nervous system. Most double-mutant mice die around 12 days of age from severe brain atrophy associated with selective neuronal loss in the cerebellar Purkinje and granule cell layers (Felbor et al. 2002), while the individual CtsB/ and CtsL/ mice are viable with normal life expectancy. If the CtsB/; CtsL/ mice were carefully nursed, the authors could extend their lifespan to an endpoint of 50 days, and analysis at that stage indicated further neuronal loss and marked atrophy in the cerebral cortex (Felbor et al. 2002). This result suggests that there is some functional overlap between certain cysteine cathepsins in performing specific physiological roles, and demonstrates the combined importance of cathepsins B and L in maintain- ing normal brain function. This functional redundancy was confirmed by rescue of the neuronal defects in CtsB–/–; CtsL–/– mice following restoration of cathepsin L by transgenic expression (Sevenich et al. 2006). Double mutants of other cathepsin genes have been generated: CtsK–/–; CtsL–/– mice (Friedrichs et al. 2003), CtsF–/–; CtsL–/– mice (Tang et al. 2006), CtsF–/–; CtsS–/– mice (Tang et al. 2006), CtsL–/–; CtsS–/– mice (Mallen-St Clair et al. 2006), and CtsB/; CtsS/ mice (Garfall et al. unpublished data), which are all viable, without any obvious additional phenotype compared to the individual knockouts, at least in the analyses that have been performed to date. Thus, whether compensatory mechanisms are activated could 296 T. Reinheckel et al. depend on several parameters, including the cell type, tissue type, and the physio- logical or pathological process involved, as well as the normal function of the particular cysteine cathepsin that is deleted.

Stromal Contributions of Cysteine Cathepsins to Tumor Development

In addition to an increase in the expression of proteolytic enzymes by tumor cells over the course of cancer progression, coopted stromal cells within the tumor microenvironment can also produce proteases. In fact, in many cancers, the stromal contribution of matrix-degrading enzymes is significantly greater than that from the transformed cells (Liotta and Kohn 2001, Jedeszko and Sloane 2004). For example, in the RT2 model, flow cytometry was used to sort whole tumors into the three major constituent cell types: endothelial, tumor, and innate immune cells (Joyce et al. 2004). When the purified cell lysates were profiled for cysteine cathepsin expression and activity, it was determined that while tumor cells preferentially expressed cathepsin L, the majority of cathepsin expression and activity was derived from the infiltrating innate immune cells (Joyce et al. 2004), namely, tumor-associated macrophages (TAMs). Indeed in certain cancers, TAMs appear to promote tumorigenesis (reviewed in Gadea and Joyce 2006); one possible mechanism could involve supplying tumor-promoting matrix-degrading enzymes, such as cathepsins B and S. In fact, cathepsin B is also expressed by macrophages in the PyMT model, where host-derived cathepsin B was shown to be more critical than tumor-derived cathepsin B in promoting lung metastasis (Vasiljeva et al. 2006). However, whether cysteine cathepsin expression from the host cells alone is generally sufficient to promote tumorigenesis in these mouse models, and more importantly in human cancer, remains an area of active research.

Conclusions and Future Directions

To date, our investigation of mouse models of human cancers with deficiencies in individual cysteine cathepsins have revealed highly context-specific functions for these proteases. In most instances, deletion of cysteine cathepsins results in reduced tumor growth and metastasis; however, the example of the protective role of cathepsin L in K14-HPV16-induced skin cancers calls for detailed investigation of additional cysteine cathepsin family members in a variety of mouse tumor models. This is even more important with regard to the discussion on the use of specific versus broad-spectrum cysteine cathepsin inhibitors as a therapeutic ap- proach for treating different malignancies including cancer (Palermo and Joyce 2008). A second conclusion from our studies is the concept that the function of cysteine cathepsins in tumorigenesis is not restricted to the cancer cell itself. 15 Roles of Cysteine Proteases in Tumor Progression 297

Rather, cysteine cathepsins appear to be highly instrumental for the activation of the tumor stroma, in which tumor-associated inflammatory cells, such as macrophages, contribute cathepsin activity with tumorigenic and prometastatic effects. A major goal in the next several years will be to identify the cell types within the microen- vironment that supply the relevant cathepsins and to determine the mechanisms by which cysteine cathepsins exert their effects on the tumor through the identification of novel substrates. Finally, it will be essential to further study the interactions between cysteine cathepsin family members and their inhibitors, as well as their interplay with other proteases, in order to identify the key proteolytic pathways critical for tumor progression and metastasis.

Acknowledgements The authors thank Eva Schill-Wendt for preparation of the illustrations in Fig. 15.1. The work of TR and CP was supported by a grant of the European Union Framework Programme 6, Project LSHC-CT-2003-503297, CancerDegradome, by grant of the Deutsche Krebshilfe (Re106977), and grant 23-7532.22-33-11/1 of the Ministerium fuer Wissenschaft und Kunst, Baden-Wuerttemberg. Research in JAJ’s laboratory was supported in part by the following: NIH R01 CA125162, NIH U54 CA125518, Sidney Kimmel Foundation for Cancer Research, V Foundation for Cancer Research, Rita Allen Foundation, Emerald Foundation, and the Geoffrey Beene Foundation. VG is the recipient of a Frank L. Horsfall Fellowship.

Reference

Adachi, W., Kawamoto, S., Ohno, I., Nishida, K., Kinoshita, S., Matsubara, K., and Okubo, K. 1998. Isolation and characterization of human : a major proteinase in corneal epithelium. Invest Ophthalmol Vis Sci 39(10): 1789–1796. Almholt, K., Lund, L.R., Rygaard, J., Nielsen, B.S., Dano, K., Romer, J., and Johnsen, M. 2005. Reduced metastasis of transgenic mammary cancer in urokinase-deficient mice. Int J Cancer 113(4): 525–532. Arbeit, J.M., Munger, K., Howley, P.M., and Hanahan, D. 1994. Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice. J Virol 68(7): 4358–4368. Authier, F., Kouach, M., and Briand, G. 2005. Endosomal proteolysis of insulin-like growth factor-I at its C-terminal D-domain by cathepsin B. FEBS Lett 579(20): 4309–4316. Bell-McGuinn, K.M., Garfall, A.L., Bogyo, M., Hanahan, D., and Joyce, J.A. 2007. Inhibition of cysteine cathepsin protease activity enhances chemotherapy regimens by decreasing tumor growth and invasiveness in a mouse model of multistage cancer. Cancer Res 67(15): 7378– 7385. Bervar, A., Zajc, I., Sever, N., Katunuma, N., Sloane, B.F., and Lah, T.T. 2003. Invasiveness of transformed human breast epithelial cell lines is related to cathepsin B and inhibited by cysteine proteinase inhibitors. Biol Chem 384(3): 447–455. Brown, J., Matutes, E., Singleton, A., Price, C., Molgaard, H., Buttle, D., and Enver, T. 1998. Lymphopain, a cytotoxic T and natural killer cell-associated cysteine proteinase. Leukemia 12 (11): 1771–1781. Buck, M.R., Karustis, D.G., Day, N.A., Honn, K.V., and Sloane, B.F. 1992. Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues. Biochem J 282(Pt 1): 273–278. 298 T. Reinheckel et al.

Carmeliet, P., and Jain, R.K. 2000. Angiogenesis in cancer and other diseases. Nature 407(6801): 249–257. Castiglioni, T., Merino, M.J., Elsner, B., Lah, T.T., Sloane, B.F., and Emmert-Buck, M.R. 1994. Immunohistochemical analysis of cathepsins D, B, and L in human breast cancer. Hum Pathol 25(9): 857–862. Cavallaro, U., and Christofori, G. 2004. Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 4(2): 118–132. Cheng, T., Hitomi, K., van Vlijmen-Willems, I.M., de Jongh, G.J., Yamamoto, K., Nishi, K., Watts, C., Reinheckel, T., Schalkwijk, J., and Zeeuwen, P.L. 2006. Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-. A novel clue for the role of cystatin M/E in epidermal cornification. J Biol Chem 281(23): 15893–15899. Coulombe, R., Grochulski, P., Sivaraman, J., Menard, R., Mort, J.S., and Cygler, M. 1996. Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment. Embo J 15(20): 5492–5503. Coussens, L.M., Hanahan, D., and Arbeit, J.M. 1996. Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice. Am J Pathol 149(6): 1899–1917. Cygler, M., Sivaraman, J., Grochulski, P., Coulombe, R., Storer, A.C., and Mort, J.S. 1996. Structure of rat procathepsin B: model for inhibition of activity by the proregion. Structure 4(4): 405–416. de Visser, K.E., Eichten, A., and Coussens, L.M. 2006. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 6(1): 24–37. Deussing, J., Kouadio, M., Rehman, S., Werber, I., Schwinde, A., and Peters, C. 2002. Identifica- tion and characterization of a dense cluster of placenta-specific cysteine peptidase genes and related genes on mouse 13. Genomics 79(2): 225–240. Drake, F.H., Dodds, R.A., James, I.E., Connor, J.R., Debouck, C., Richardson, S., Lee-Rykac- zewski, E., Coleman, L., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M. 1996. Cathepsin K, but not cathepsins B, L, or S, is abundantly expressed in human osteoclasts. J Biol Chem 271(21): 12511–12516. Ebert, M.P., Kruger, S., Fogeron, M.L., Lamer, S., Chen, J., Pross, M., Schulz, H.U., Lage, H., Heim, S., Roessner, A., Malfertheiner, P., and Rocken, C. 2005. Overexpression of cathepsin B in gastric cancer identified by proteome analysis. Proteomics 5(6): 1693–1704. Eeckhout, Y., and Vaes, G. 1977. Further studies on the activation of procollagenase, the latent precursor of bone collagenase. Effects of lysosomal cathepsin B, plasmin and kallikrein, and spontaneous activation. Biochem J 166(1): 21–31. Felbor, U., Dreier, L., Bryant, R.A., Ploegh, H.L., Olsen, B.R., and Mothes, W. 2000. Secreted cathepsin L generates endostatin from collagen XVIII. Embo J 19(6): 1187–1194. Felbor, U., Kessler, B., Mothes, W., Goebel, H.H., Ploegh, H.L., Bronson, R.T., and Olsen, B.R. 2002. Neuronal loss and brain atrophy in mice lacking cathepsins B and L. Proc Natl Acad Sci USA99(12): 7883–7888. Ferreras, M., Felbor, U., Lenhard, T., Olsen, B.R., and Delaisse, J. 2000. Generation and degrada- tion of human endostatin proteins by various proteinases. FEBS Lett 486(3): 247–251. Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M., Elling, F., Leist, M., and Jaattela, M. 2001. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 153(5): 999–1010. Friedrichs, B., Tepel, C., Reinheckel, T., Deussing J., von Figura, K., Herzog, V., Peter, C., Saftig, P., Brix, K., 2003. Thyroid functions of mouse cathepsins B, K, and L. J Clin Invest. 111 (11): 1733–1745. Frohlich, E., Schlagenhauff, B., Mohrle, M., Weber, E., Klessen, C., and Rassner, G. 2001. Activity, expression, and transcription rate of the cathepsins B, D, H, and L in cutaneous malignant melanoma. Cancer 91(5): 972–982. Fujise, N., Nanashim, A., Taniguchi, Y., Matsuo, S., Hatano, K., Matsumoto, Y., Tagawa, Y., and Ayabe, H. 2000. Prognostic impact of cathepsin B and matrix metalloproteinase-9 in pulmo- nary adenocarcinomas by immunohistochemical study. Lung Cancer 27(1): 19–26. 15 Roles of Cysteine Proteases in Tumor Progression 299

Gadea, B.B., and Joyce, J.A. 2006. Tumour-host interactions: implications for developing anti- cancer therapies. Expert Rev Mol Med 8(30): 1–32. Gocheva, V., and Joyce, J.A. 2007. Cysteine cathepsins and the cutting edge of cancer invasion. Cell Cycle 6(1): 60–64. Gocheva, V., Zeng, W., Ke, D., Klimstra, D., Reinheckel, T., Peters, C., Hanahan, D., and Joyce, J. A. 2006. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis. Genes Dev 20 (5): 543–556. Goretzki, L., Schmitt, M., Mann, K., Calvete, J., Chucholowski, N., Kramer, M., Gunzler, W.A., Janicke, F., and Graeff, H. 1992. Effective activation of the proenzyme form of the urokinase- type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L. FEBS Lett 297(1–2): 112–118. Goulet, B., Baruch, A., Moon, N.S., Poirier, M., Sansregret, L.L., Erickson, A., Bogyo, M., and Nepveu, A. 2004. A cathepsin L isoform that is devoid of a signal peptide localizes to the nucleus in S phase and processes the CDP/Cux transcription factor. Mol Cell 14(2): 207–219. Guicciardi, M.E., Deussing, J., Miyoshi, H., Bronk, S.F., Svingen, P.A., Peters, C., Kaufmann, S. H., and Gores, G.J. 2000. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apopto- sis by promoting mitochondrial release of cytochrome c. J Clin Invest 106(9): 1127–1137. Guicciardi, M.E., Miyoshi, H., Bronk, S.F., and Gores, G.J. 2001. Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: impli- cations for therapeutic applications. Am J Pathol 159(6): 2045–2054. Guinec, N., Dalet-Fumeron, V., and Pagano, M. 1993. ‘‘In vitro’’ study of basement membrane degradation by the cysteine proteinases, cathepsins B, B-like and L. Digestion of collagen IV, laminin, fibronectin, and release of gelatinase activities from basement membrane fibronectin. Biol Chem Hoppe Seyler 374(12): 1135–1146. Guo, M., Mathieu, P.A., Linebaugh, B., Sloane, B.F., and Reiners, J.J., Jr. 2002. Phorbol ester activation of a proteolytic cascade capable of activating latent transforming growth factor- betaL a process initiated by the exocytosis of cathepsin B. J Biol Chem 277(17): 14829–14837. Guy, C.T., Cardiff, R.D., and Muller, W.J. 1992. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol 12(3): 954–961. Halangk, W., Lerch, M.M., Brandt-Nedelev, B., Roth, W., Ruthenbuerger, M., Reinheckel, T., Domschke, W., Lippert, H., Peters, C., and Deussing, J. 2000. Role of cathepsin B in intracel- lular trypsinogen activation and the onset of acute pancreatitis. J Clin Invest 106(6): 773–781. Hanahan, D. 1985. Heritable formation of pancreatic beta-cell tumours in transgenic mice expres- sing recombinant insulin/simian virus 40 oncogenes. Nature 315(6015): 115–122. Hanahan, D., and Folkman, J. 1996. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86(3): 353–364. Harbeck, N., Alt, U., Berger, U., Kruger, A., Thomssen, C., Janicke, F., Hofler, H., Kates, R.E., and Schmitt, M. 2001. Prognostic impact of proteolytic factors (urokinase-type plasminogen activator, plasminogen activator inhibitor 1, and cathepsins B, D, and L) in primary breast cancer reflects effects of adjuvant systemic therapy. Clin Cancer Res 7(9): 2757–2764. Ishidoh, K., and Kominami, E. 1995. Procathepsin L degrades extracellular matrix proteins in the presence of glycosaminoglycans in vitro. Biochem Biophys Res Commun 217(2): 624–631. Jedeszko, C., and Sloane, B.F. 2004. Cysteine cathepsins in human cancer. Biol Chem 385(11): 1017–1027. Joyce, J.A., Baruch, A., Chehade, K., Meyer-Morse, N., Giraudo, E., Tsai, F.Y., Greenbaum, D.C., Hager, J.H., Bogyo, M., and Hanahan, D. 2004. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell 5(5): 443–453. Kopitar-Jerala, N. 2006. The role of cystatins in cells of the immune system. FEBS Lett 580(27): 6295–6301. Kos, J., Stabuc, B., Schweiger, A., Krasovec, M., Cimerman, N., Kopitar-Jerala, N., and Vrhovec, I. 1997. Cathepsins B, H, and L and their inhibitors stefin A and cystatin C in sera of melanoma patients. Clin Cancer Res 3(10): 1815–1822. 300 T. Reinheckel et al.

Lah, T.T., Buck, M.R., Honn, K.V., Crissman, J.D., Rao, N.C., Liotta, L.A., and Sloane, B.F. 1989. Degradation of laminin by human tumor cathepsin B. Clin Exp Metastasis 7(4): 461–468. Lah, T.T., Cercek, M., Blejec, A., Kos, J., Gorodetsky, E., Somers, R., and Daskal, I. 2000. Cathepsin B, a prognostic indicator in lymph node-negative breast carcinoma patients: com- parison with cathepsin D, cathepsin L, and other clinical indicators. Clin Cancer Res 6(2): 578– 584. Lecaille, F., Kaleta, J., and Bromme, D. 2002. Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design. Chem Rev 102(12): 4459–4488. Lechner, A.M., Assfalg-Machleidt, I., Zahler, S., Stoeckelhuber, M., Machleidt, W., Jochum, M., and Nagler, D.K. 2006. RGD-dependent binding of procathepsin X to integrin alphavbeta3 mediates cell-adhesive properties. J Biol Chem 281(51): 39588–39597. Liotta, L.A., and Kohn, E.C. 2001. The microenvironment of the tumour-host interface. Nature 411(6835): 375–379. Liu, Y., Xiao, S., Shi, Y., Wang, L., Ren, W., and Sloane, B.F. 1998. Cathepsin B on invasion and metastasis of gastric carcinoma. Chin Med J (Engl) 111(9): 784–788. Lopez, T., and Hanahan, D. 2002. Elevated levels of IGF-1 receptor convey invasive and metastatic capability in a mouse model of pancreatic islet tumorigenesis. Cancer Cell 1(4): 339–353. Mai, J., Finley, R.L., Jr., Waisman, D.M., and Sloane, B.F. 2000. Human procathepsin B interacts with the annexin II tetramer on the surface of tumor cells. J Biol Chem 275(17): 12806–12812. Mai, J., Sameni, M., Mikkelsen, T., and Sloane, B.F. 2002. Degradation of extracellular matrix protein tenascin-C by cathepsin B: an interaction involved in the progression of gliomas. Biol Chem 383(9): 1407–1413. Mallen-St Clair, J., Shi, G.P., Sutherland, R.E., Chapman, H.A., Caughey, G.H., and Wolters, P.J. 2006. Cathepsins L and S are not required for activation of dipeptidyl peptidase I (cathepsin C) in mice. Biol Chem 387(8): 1143–1146. Mason, R.W., Gal, S., and Gottesman, M.M. 1987. The identification of the major excreted protein (MEP) from a transformed mouse fibroblast cell line as a catalytically active precursor form of cathepsin L. Biochem J 248(2): 449–454. Miyake, H., Hara, I., and Eto, H. 2004. Serum level of cathepsin B and its density in men with prostate cancer as novel markers of disease progression. Anticancer Res 24(4): 2573–2577. Mohamed, M.M., and Sloane, B.F. 2006. Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer 6(10): 764–775. Mort, J.S., Recklies, A.D., and Poole, A.R. 1985. Release of cathepsin B precursors from human and murine tumours. Prog Clin Biol Res 180: 243–245. Nagler, D.K., Kruger, S., Kellner, A., Ziomek, E., Menard, R., Buhtz, P., Krams, M., Roessner, A., and Kellner, U. 2004. Up-regulation of cathepsin X in prostate cancer and prostatic intrae- pithelial neoplasia. Prostate 60(2): 109–119. Nasu, K., Kai, K., Fujisawa, K., Takai, N., Nishida, Y., and Miyakawa, I. 2001. Expression of cathepsin L in normal endometrium and endometrial cancer. Eur J Obstet Gynecol Reprod Biol 99(1): 102–105. Nolan, D.J., Ciarrocchi, A., Mellick, A.S., Jaggi, J.S., Bambino, K., Gupta, S., Heikamp, E., McDevitt, M.R., Scheinberg, D.A., Benezra, R., and Mittal, V. 2007. Bone marrow-derived endothelial progenitor cells are a major determinant of nascent tumor neovascularization. Genes Dev 21(12): 1546–1558. Ondr, J.K., and Pham, C.T. 2004. Characterization of murine cathepsin W and its role in cell- mediated cytotoxicity. J Biol Chem 279 (26): 27525–27533. Palermo, C., and Joyce, J.A. 2008. Cysteine cathepsin proteases as pharmacological targets in cancer. Trends Pharmacol Sci 29(1):22–28. Perl, A.K., Wilgenbus, P., Dahl, U., Semb, H., and Christofori, G. 1998. A causal role for E- cadherin in the transition from adenoma to carcinoma. Nature 392(6672): 190–193. 15 Roles of Cysteine Proteases in Tumor Progression 301

Petermann, I., Mayer, C., Stypmann, J., Biniossek, M.L., Tobin, D.J., Engelen, M.A., Dandekar, T., Grune, T., Schild, L., Peters, C., and Reinheckel, T. 2006. Lysosomal, cytoskeletal, and metabolic alterations in cardiomyopathy of cathepsin L knockout mice. Faseb J 20(8): 1266– 1268. Pham, C.T., and Ley, T.J. 1999. Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo. Proc Natl Acad Sci U S A 96(15): 8627–8632. Poole, A.R., Tiltman, K.J., Recklies, A.D., and Stoker, T.A. 1978. Differences in secretion of the proteinase cathepsin B at the edges of human breast carcinomas and fibroadenomas. Nature 273(5663): 545–547. Rao, J.S. 2003. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer 3(7): 489–501. Reinheckel, T., Hagemann, S., Dollwet-Mack, S., Martinez, E., Lohmuller, T., Zlatkovic, G., Tobin, D.J., Maas-Szabowski, N., and Peters, C. 2005. The lysosomal cysteine protease cathepsin L regulates keratinocyte proliferation by control of growth factor recycling. J Cell Sci 118(Pt 15): 3387–3395. Rempel, S.A., Rosenblum, M.L., Mikkelsen, T., Yan, P.S., Ellis, K.D., Golembieski, W.A., Sameni, M., Rozhin, J., Ziegler, G., and Sloane, B.F. 1994. Cathepsin B expression and localization in glioma progression and invasion. Cancer Res 54(23): 6027–6031. Roth, W., Deussing, J., Botchkarev, V.A., Pauly-Evers, M., Saftig, P., Hafner, A., Schmidt, P., Schmahl, W., Scherer, J., Anton-Lamprecht, I., Von Figura, K., Paus, R., and Peters, C. 2000. Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling. Faseb J 14(13): 2075–2086. Saad, Z., Bramwell, V.H., Wilson, S.M., O’Malley, F.P., Jeacock, J., and Chambers, A.F. 1998. Expression of genes that contribute to proliferative and metastatic ability in breast cancer resected during various menstrual phases. Lancet 351(9110): 1170–1173. Saftig, P., Hunziker, E., Wehmeyer, O., Jones, S., Boyde, A., Rommerskirch, W., Moritz, J.D., Schu, P., and von Figura, K. 1998. Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci U S A 95(23): 13453–13458. Sameni, M., Moin, K., and Sloane, B.F. 2000. Imaging proteolysis by living human breast cancer cells. Neoplasia 2(6): 496–504. Sameni, M., Dosescu, J., and Sloane, B.F. 2001. Imaging proteolysis by living human glioma cells. Biol Chem 382(5): 785–788. Santamaria, I., Velasco, G., Cazorla, M., Fueyo, A., Campo, E., and Lopez-Otin, C. 1998. Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcino- mas. Cancer Res 58(8): 1624–1630. Schweiger, A., Staib, A., Werle, B., Krasovec, M., Lah, T.T., Ebert, W., Turk, V., and Kos, J. 2000. Cysteine proteinase cathepsin H in tumours and sera of lung cancer patients: relation to prognosis and cigarette smoking. Br J Cancer 82(4): 782–788. Sevenich, L., Pennacchio, L.A., Peters, C., and Reinheckel, T. 2006. Human cathepsin L rescues the neurodegeneration and lethality in cathepsin B/L double-deficient mice. Biol Chem 387(7): 885–891. Shi, G.P., Webb, A.C., Foster, K.E., Knoll, J.H., Lemere, C.A., Munger, J.S., and Chapman, H.A. 1994. Human cathepsin S: chromosomal localization, gene structure, and tissue distribution. J Biol Chem 269(15): 11530–11536. Shi, G.P., Villadangos, J.A., Dranoff, G., Small, C., Gu, L., Haley, K.J., Riese, R., Ploegh, H.L., and Chapman, H.A. 1999. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity 10(2): 197–206. Shi, G.P., Sukhova, G.K., Kuzuya, M., Ye, Q., Du, J., Zhang, Y., Pan, J.H., Lu, M.L., Cheng, X. W., Iguchi, A., Perrey, S., Lee, A.M., Chapman, H.A., and Libby, P. 2003. Deficiency of the cysteine protease cathepsin S impairs microvessel growth. Circ Res 92(5): 493–500. Sinha, A.A., Gleason, D.F., Staley, N.A., Wilson, M.J., Sameni, M., and Sloane, B.F. 1995. Cathepsin B in angiogenesis of human prostate: an immunohistochemical and immunoelectron microscopic analysis. Anat Rec 241(3): 353–362. 302 T. Reinheckel et al.

Sivaraman, J., Lalumiere, M., Menard, R., and Cygler, M. 1999. Crystal structure of wild-type human procathepsin K. Protein Sci 8(2): 283–290. Strojnik, T., Kos, J., Zidanik, B., Golouh, R., and Lah, T. 1999. Cathepsin B immunohistochemical staining in tumor and endothelial cells is a new prognostic factor for survival in patients with brain tumors. Clin Cancer Res 5(3): 559–567. Stypmann, J., Glaser, K., Roth, W., Tobin, D.J., Petermann, I., Matthias, R., Monnig, G., Haverkamp, W., Breithardt, G., Schmahl, W., Peters, C., and Reinheckel, T. 2002. Dilated cardiomyopathy in mice deficient for the lysosomal cysteine peptidase cathepsin L. Proc Natl Acad Sci USA 99(9): 6234–6239. Tang, C.H., Lee, J.W., Galvez, M.G., Robillard, L., Mole, S.E., and Chapman, H.A. 2006. Murine cathepsin f deficiency causes neuronal lipofuscinosis and late-onset neurological disease. Mol Cell Biol 26(6): 2309–2316. Thomssen, C., Schmitt, M., Goretzki, L., Oppelt, P., Pache, L., Dettmar, P., Janicke, F., and Graeff, H. 1995. Prognostic value of the cysteine proteases cathepsins B and cathepsin L in human breast cancer. Clin Cancer Res 1(7): 741–746. Turk, B., Turk, D., and Turk, V. 2000. Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta 1477(1–2): 98–111. Turk, V., Turk, B., and Turk, D. 2001. Lysosomal cysteine proteases: facts and opportunities. Embo J 20(17): 4629–4633. Urbich, C., Heeschen, C., Aicher, A., Sasaki, K., Bruhl, T., Farhadi, M.R., Vajkoczy, P., Hofmann, W.K., Peters, C., Pennacchio, L.A., Abolmaali, N.D., Chavakis, E., Reinheckel, T., Zeiher, A. M., and Dimmeler, S. 2005. Cathepsin L is required for endothelial progenitor cell-induced neovascularization. Nat Med 11(2): 206–213. Vasiljeva, O., Dolinar, M., Pungercar, J.R., Turk, V., and Turk, B. 2005. Recombinant human procathepsin S is capable of autocatalytic processing at neutral pH in the presence of glyco- saminoglycans. FEBS Lett 579(5): 1285–1290. Vasiljeva, O., Papazoglou, A., Kruger, A., Brodoefel, H., Korovin, M., Deussing, J., Augustin, N., Nielsen, B.S., Almholt, K., Bogyo, M., Peters, C., and Reinheckel, T. 2006. Tumor cell-derived and macrophage-derived cathepsin B promotes progression and lung metastasis of mammary cancer. Cancer Res 66(10): 5242–5250. Wang, B., Sun, J., Kitamoto, S., Yang, M., Grubb, A., Chapman, H.A., Kalluri, R., and Shi, G.P. 2006. Cathepsin S controls angiogenesis and tumor growth via matrix-derived angiogenic factors. J Biol Chem 281(9): 6020–6029. Watanabe, M., Higashi, T., Watanabe, A., Osawa, T., Sato, Y., Kimura, Y., Tominaga, S., Hashimoto, N., Yoshida, Y., Morimoto, S., and et al. 1989. Cathepsin B and L activities in gastric cancer tissue: correlation with histological findings. Biochem Med Metab Biol 42(1): 21–29. Werle, B., Lotterle, H., Schanzenbacher, U., Lah, T.T., Kalman, E., Kayser, K., Bulzebruck, H., Schirren, J., Krasovec, M., Kos, J., and Spiess, E. 1999. Immunochemical analysis of cathepsin B in lung tumours: an independent prognostic factor for squamous cell carcinoma patients. Br J Cancer 81(3): 510–519. Wiederanders, B., and Kirschke, H. 1989. The processing of a cathepsin L precursor in vitro. Arch Biochem Biophys 272(2): 516–521. Yasuda, Y., Li, Z., Greenbaum, D., Bogyo, M., Weber, E., and Bromme, D. 2004. Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages. J Biol Chem 279(35): 36761–36770. zur Hausen, H. 1987. Papillomaviruses in human cancer. Cancer 59(10): 1692–1696.