Oncogene (2010) 29, 1611–1621 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Deficiency for the cysteine protease cathepsin L promotes tumor progression in mouse epidermis J Dennema¨rker1,6, T Lohmu¨ller1,6, J Mayerle2, M Tacke1, MM Lerch2, LM Coussens3,4, C Peters1,5 and T Reinheckel1,5 1Institute for Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Freiburg, Germany; 2Department of Gastroenterology, Endocrinology and Nutrition, Ernst-Moritz-Arndt University Greifswald, Greifswald, Germany; 3Department of Pathology, University of California, San Francisco, CA, USA; 4Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA and 5Ludwig Heilmeyer Comprehensive Cancer Center and Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Freiburg, Germany To define a functional role for the endosomal/lysosomal Introduction cysteine protease cathepsin L (Ctsl) during squamous carcinogenesis, we generated mice harboring a constitutive Proteases have traditionally been thought to promote Ctsl deficiency in addition to epithelial expression of the invasive growth of carcinomas, contributing to the the human papillomavirus type 16 oncogenes (human spread and homing of metastasizing cancer cells (Dano cytokeratin 14 (K14)–HPV16). We found enhanced tumor et al., 1999). Proteases that function outside tumor cells progression and metastasis in the absence of Ctsl. As have been implicated in these processes because of their tumor progression in K14–HPV16 mice is dependent well-established release from tumors, causing the break- on inflammation and angiogenesis, we examined immune down of basement membranes and extracellular matrix. cell infiltration and vascularization without finding any Thus, extracellular proteases may in part facilitate effect of the Ctsl genotype. In contrast, keratinocyte- tumor cell invasion into ectopic tissue by the generation specific transgenic expression of cathepsin V, the human of bioactive factors from extracellular matrix macro- orthologue of mouse Ctsl, in otherwise Ctsl-deficient K14– molecules (Kalluri, 2003; DeClerck et al., 2004). The HPV16 mice restored the phenotype observed in the groups of tumor-promoting proteolytic enzymes consist control HPV16 skin. To better understand this phenotype of: soluble and integral membrane matrix metallopro- at the molecular level, we measured several oncogenic teases (MMP, MT-MMP, and ADAMTS), serine signal transduction pathways in primary keratinocytes on proteases and inhibitors of the plasminogen activator/ stimulation with keratinocyte-conditioned cell culture plasminogen system, and cysteine- and aspartyl-type medium. We found increased activation of protein kinase endosomal/lysosomal peptidases (Liotta and Kohn, B/Akt and mitogen-activated protein kinase pathways in 2001; Mohamed and Sloane, 2006). Alternatively, a protease-deficient cells, especially if treated with media number of proteases are considered to have tumor- conditioned by Ctsl-deficient keratinocytes. Similarly, the suppressive functions (Lopez-Otin and Matrisian, 2007). level of active GTP-Ras was increased in Ctsl-deficient This paradox is easily appreciated for caspases that epidermis. We conclude that Ctsl is critical for the regulate cell death programming in cells through termination of growth factor signaling in the endosomal/ initiation and execution of apoptotic programs. How- lysosomal compartment of keratinocytes and, therefore, ever, there is also evidence of anti-tumor functions for functions as an anti-tumor protease. some members of the extracellular metalloprotease Oncogene (2010) 29, 1611–1621; doi:10.1038/onc.2009.466; families, including MMPs and ADAMTS (Martin and published online 21 December 2009 Matrisian, 2007). The major mechanism(s) underlying anti-tumor functions is thought to result from the Keywords: skin cancer; mouse model; protease; inhibition of tumor angiogenesis because of the genera- cathepsin tion of anti-angiogenic peptides (Nyberg et al., 2008). Cathepsin L (Ctsl) is an endosomal/lysosomal papain- like cysteine endoprotease implicated in a variety of physiological and pathological processes (Vasiljeva et al., 2007). The mouse genome encodes for only one Correspondence: Dr T Reinheckel, Institut fu¨r Molekulare Medizin Ctsl while in humans there is not only the ‘classical’ und Zellforschung, Albert-Ludwigs-University, Stefan Meier Strasse cathepsin L (CTSL) but also the related cysteine 17, Freiburg, D-79104, Germany. protease cathepsin V (CTSV), which is frequently E-mail: [email protected] 6These authors contributed equally to this work. termed cathepsin L2. Human CTSV and murine Ctsl Received 1 July 2009; revised 7 October 2009; accepted 16 November are true orthologues because they share 75% amino 2009; published online 21 December 2009 acid sequence identity, whereas the homology of murine Cathepsin L in skin cancer J Dennema¨rker et al 1612 Ctsl and human CTSL is less pronounced (Bro¨mme, Results 2004). However, human CTSL is ubiquitously ex- pressed, whereas CTSV expression is restricted to Ctsl deficiency potentiates neoplastic progression macrophages, thymus, testis, corneal epithelium and— in K14–HPV16 transgenic mice importantly—the epidermis (Bernard et al., 2003; Cysteine cathepsins, such as Ctsl, are considered to be Tolosa et al., 2003; Cheng et al., 2006). In a variety of multifunctional proteases in cancer (Mohamed human cancers expression levels of CTSL have been and Sloane, 2006). To evaluate which aspects of positively correlated with poor prognosis (Jedeszko and neoplastic progression Ctsl might regulate, we back- Sloane, 2004), while CTSV is transcriptionally upregu- crossed CtslÀ/À mice (Roth et al., 2000) into the FVB/n lated in skin squamous cell carcinomas (SCCs) as genetic background and crossed them to congenic compared with the benign hyperproliferation in psor- K14–HPV16 (HPV16) mice. We found that HPV16/ iatic skin (Haider et al., 2006). In addition to their CtslÀ/À mice developed the first palpable tumors intracellular localization, the various human and murine significantly earlier than HPV16/Ctsl þ /À and HPV16/ Ctsl enzymes have been detected in the extracellular Ctsl þ / þ littermates (Figure 1a). The final tumor size of portion of the plasma membrane and secreted into the 1cm3 appeared in the HPV16/CtslÀ/À mice approxi- extracellular space. Ctsl released by carcinoma cells has mately 10 weeks earlier than in the controls (Figure 1b). been shown to produce the angiogenesis inhibitor In addition, histopathological grading of carcinomas endostatin from collagen XVIII, the core protein of from HPV16/CtslÀ/À mice revealed the emergence of heparan sulfate proteoglycans in vascular and epithelial only poorly differentiated grade III and grade IV SCCs basement membranes (Felbor et al., 2000). In contrast, as opposed to a higher percentage of well- and Urbich et al. showed that Ctsl is actually critical for the moderately differentiated SCCs in littermate controls invasive capacity of endothelial progenitor cells, pro- (Figure 1c). To further assess the histological character- moting neovascularization (Urbich et al., 2005). Intra- istics of carcinomas, we quantified membrane-bound cellular Ctsl has been identified as a positive regulator of E-cadherin in grade III SCCs from HPV16/Ctsl þ / þ and insulin-like growth factor 1 receptor signaling and of the HPV16/CtslÀ/À mice (Figure 1d). We found that experimental metastasis of tumor cells, which are membrane-bound E-cadherin was reduced in the dependent on insulin-like growth factor 1 receptor, Ctsl-deficient carcinomas, substantiating their poor and has also been found to have a role in promoting histological differentiation and suggesting a highly resistance toward chemotherapeutic drugs (Zheng et al., invasive and potentially metastatic phenotype. Accord- 2004; Navab et al., 2008). N-terminally truncated Ctsl ingly, the frequency of metastases in axillary lymph has been found in the nucleus, in which it activates the nodes was found to be significantly higher in the Ctsl- CDP/Cux transcription factor, a positive regulator of deficient mice, 25% in the HPV16/Ctsl þ / þ cohort and the cell cycle involved in cellular transformation (Goulet 70% in HPV16/CtslÀ/À mice (Figure 1e). The early onset et al., 2007). and poor state of differentiation in HPV16/CtslÀ/À As of the multiple potential mechanisms by which cancers was preceded by an earlier development Ctsl might affect tumor progression, we decided of dysplasias (Figure 2a). Epidermal dysplasia was to analyze this protease in a complex in vivo model found at 16 weeks of age in each of the HPV16/CtslÀ/À of tumorigenesis that mimics the successive stages mice, whereas only 50% of control mice had advanced of human cancer development. Therefore, we used to the dysplastic state by this time point. Quantification K14–HPV16 transgenic mice as a mouse model of of the cell proliferation marker Ki67 revealed signifi- epidermal carcinogenesis (Arbeit et al., 1994; Coussens cantly increased percentages of proliferating keratino- et al., 1996). K14–HPV16 mice express the early cytes in the premalignant epidermis of HPV16/CtslÀ/À region genes of human papillomavirus type 16 mice at 8, 16 and 24 weeks of age (Figures 2b and c). (HPV16) under the control of the human cytokeratin Together, these data indicate a potential anti-tumor role 14 (K14) promoter. HPV16 transgenic mice show a for Ctsl