The lysosomal cysteine proteases cathepsin B (Ctsb) and cathepsin Z (Ctsz, also called cathepsin X/P) have been implicated in cancer pathogenesis. Compensation of Ctsb by Ctsz in Ctsb −/− mice has been suggested. To further define the functional interplay of these proteases in the context of cancer, we generated Ctsz null mice, crossed them with Ctsb-deficient mice harboring a transgene for the mammary duct–specific expression of polyoma middle T oncogene (PymT), and analyzed the effects of single and combined Ctsb and Ctsz deficiencies on breast cancer progression. Single Ctsb deficiency resulted in delayed detection of first tumors and reduced tumor burden, whereas Ctsz-deficient mice had only a prolonged tumor-free period. However, only a trend toward reduced metastatic burden without statistical significance was detected in both single mutants. Strikingly, combined loss of Ctsb and Ctsz led to additive effects, resulting in significant and prominent delay of early and advanced tumor development, improved histopathologic tumor grading, as well as a 70% reduction in the number of lung metastases and an 80% reduction in the size of these metastases. We conclude that the double deficiency of Ctsb and Ctsz exerts significant synergistic anticancer effects, whereas the single deficiencies demonstrate at least partial reciprocal compensation.

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In several human cancers elevated levels of lysosomal cysteine cathepsins correlate with poor prognosis. Transgenic overexpression of cathepsin B (CTSB) in the murine MMTV-PyMT model of breast cancer facilitates tumor invasion and metastasis1 whereas deficiencies of cathepsins B and Z synergistically attenuate these processes2. Notably, cathepsins are expressed in cancer cells as well as in cells of the tumor microenvironment. Our current investigations address the issue of cell-type specific functions of cathepsins B and Z in progression and metastasis of MMTV-PyMT mammary cancers by a combination of in vivo and in vitro approaches. Orthotopic implantation revealed that CTSB overexpression in cancer cells rather than in cells of the stroma affects tumor progression in this transgenic system. In 3D cultures primary PyMT cancer cells overexpressing CTSB revealed an elevated extracellular matrix degradation as well as an enhanced collective cell invasion. Co-cultivation of PyMT cancer cells with bone marrow derived macrophages induced a M2-polarized, tumor associated macrophage (TAM)-like phenotype and enhanced sprouting in tumor spheroids. A doxycycline (DOX)-inducible CTSB expression system was employed to selectively overexpress human CTSB in either cancer cells or in macrophages in 3D-cocultures. Interestingly, in this co-culture system selective overexpression of CTSB in either cancer cells or TAMs resulted in enhanced spheroid sprouting only if CTSB was overexpressed in the cancer cells. The carboxypeptidase cathepsin Z (CTSZ) is the only cysteine cathepsin harboring a RGD integrin binding motif. Expressing mutants of this enzyme revealed that the RGD motif is pro-invasive while the proteolytic activity is suppressing the invasive phenotype. The influence of the cysteine cathepsins CTSB and CTSZ on the secretome of PyMT cancer cells co-cultured with either wild-type or CTSB/CTSZ double knock macrophages was investigated by comparative proteomics. This approach revealed that the lysosomal glycoprotein CREG1 involved in cell proliferation and differentiation was significantly elevated in the secretome of the co-cultures with the double knock out macrophages. These results underline that cysteine cathepsins can have an impact on tumor invasion and metastasis on different levels, direct modification of the extracellular matrix as well as interference with putative signaling molecules. 1. Sevenich L, et al. Oncogene 2011; 30:54-64. 2. Sevenich L, et al. Proc Natl Acad Sci U S A 2010; 107: 2497-2502 Citation Format: Christoph Peters, Fee Bengsch, Kathrin Sachse, Alejandro Gomez-Auli, Oliver Schilling, Thomas Reinheckel. Cell type-dependent functions of cysteine cathepsins in murine breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B28.

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