Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice Lisa Sevenicha,b, Uta Schurigta,1, Kathrin Sachsea,b, Mieczyslaw Gajdac, Fee Wernera,b,d, Sebastian Müllera, Olga Vasiljevaa,2, Anne Schwindea, Nicole Klemma, Jan Deussinga,3, Christoph Petersa,e, and Thomas Reinheckela,e,4 a Institute of Molecular Medicine and Cell Research and bFaculty of Biology, Albert Ludwigs University, Freiburg D-79104, Germany; cInstitute of Pathology, Friedrich Schiller University, Jena D-07740, Germany; and dSpemann Graduate School of Biology and Medicine and eLudwig Heilmeyer Comprehensive Cancer Center and Center for Biological Signaling Studies, Albert Ludwigs University, Freiburg D-79106, Germany
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. lysosome
| mammary adenocarcinoma | mouse model | protease
P
roteolysis is a hallmark of invasive tumor growth and metastasis. Proteases regulate these processes through several possible mechanisms. Processing of cell-adhesion molecules (e.g., E-cadherin) disrupts cell–cell contacts, facilitating cancer cell dissemination from the primary site (1). The breakdown of the basement membrane by degradation of ECM proteins is essential for invasion into surrounding tissue or blood vessels (2). Furthermore, proteolytic events affect tumor biological processes, such as proliferation and angiogenesis, by liberating ECM-bound growth and angiogenic factors or by processing and activating these factors (3, 4). Several protease classes, including metallo-, aspartic, serine, and cysteine proteases, are implicated in promoting tumor progression and metastasis (5–8). The family of lysosomal cysteine proteases, the cysteine cathepsins (clan CA; C1 family), has recently attracted attention as tumor-promoting enzymes (9). Eleven cysteine cathepsins are annotated in the human genome: cathepsin B, C, F, H, K, L, L2/V, O, S, W, and X/ Z/P (10). Cysteine cathepsins are implicated primarily in terminal protein degradation in the acidic environment of the lysosomes (11); however, they also exhibit specific functions in physiological (12) and pathological processes (13). Despite the cathepsins’ general localization in the endosomal/lysosomal compartment, changes in distribution during neoplasia formation result in secretion and extracellular effects (14, 15). Various human malignancies show overexpression of some cysteine cathepsins and accumulation at the invasive tumor front of solid cancers (16, 17). This increased expression and proteolytic activity correlates www.pnas.org/cgi/doi/10.1073/pnas.0907240107
with poor prognosis in several human cancers, including breast cancer (18). Histological studies on human cancers and studies using ex vivo models implicate mainly cathepsin B and L, and to a lesser extent cathepsin H, S, and Z, in the proteolytic events promoting cancer progression (19). Cancer mouse models with cathepsin deficiencies allow the elucidation of specific functions of individual cathepsins in tumor progression and metastasis (1, 20). Using the transgenic polyoma middle T oncogene (PymT)-induced mouse model for breast cancer (21), we recently showed that ablation of one cathepsin B (Ctsb) allele was sufficient to significantly reduce lung metastasis (20). However, a deficiency of both Ctsb alleles did not further reduce the metastatic burden; rather, statistical significance compared with Ctsb WT mice was lost. This effect can be explained by a compensation of Ctsb deficiency by cathepsin Z (Ctsz) in primary Ctsb-deficient tumor cells derived from breast cancers of Ctsb−/− mice (20). Ctsz (also known as cathepsin X or cathepsin P) belongs to the same Clan CA/C1 protease family as Ctsb (22–24). Ctsz shows unique features among these proteases, that is, the presence of a RGD motif for integrin binding in its propeptide and strict exopeptidase activity (25, 26). Notably, Ctsb and Ctsz are the only carboxypeptidases in the cysteine cathepsin family, although Ctsb also exhibits endopeptidase activity (27, 28). To elucidate specific functions of Ctsz in cancer progression, we generated Ctszdeficient mice and bred them with transgenic mice expressing PymT under control of the mammary epithelium-specific mouse mammary tumor virus (MMTV) LTR promoter (21). We demonstrate that Ctsb and Ctsz single deficiencies have only marginal effects on most parameters of breast cancer development in this model, but that combined deficiency of Ctsb and Ctsz results in a significantly reduced tumor and metastatic burden of PymTinduced breast cancer in mice. Results Generation and Phenotyping of Mice with Ctsb/Ctsz Double Deficiency. To investigate the effects of single and combined defi-
ciencies of Ctsb and Ctsz in the context of breast cancer, we gen-
Author contributions: L.S., U.S., O.V., J.M.D., C.P., and T.R. designed research; L.S., U.S., K.S., M.G., F.W., S.M., O.V., A.S., N.K., J.M.D., and T.R. performed research; L.S., U.S., K.S., F.W., S.M., A.S., C.P., and T.R. analyzed data; and L.S., J.M.D., and T.R. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
Present address: Institute for Molecular Infection Research, D-97070 Wuerzburg, Germany.
2
Present address: Jozef Stefan Institute, Department of of Biochemistry and Molecular and Structural Biology, SI-1000 Ljubljana, Slovenia.
3
Present address: Max Planck Institute for Psychiatry, Molecular Neurogenetics, D-80804 Munich, Germany.
4
To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0907240107/DCSupplemental.
PNAS | February 9, 2010 | vol. 107 | no. 6 | 2497–2502
CELL BIOLOGY
Edited by William S. Sly, Saint Louis University School of Medicine, St. Louis, MO, and approved December 22, 2009 (received for review June 29, 2009)
erated Ctsz-deficient mice (Ctsz−/−) and backcrossed them to the FVB/N genetic background (SI Methods and Fig. S1). We then further crossed these mice with congenic Ctsb-deficient (Ctsb−/−) mice harboring a construct for expression of PymT in mammary epithelial cells under control of the MMTV LTR promoter (20, 21). Female mice hemizygous for the PymT transgene were grouped according to their Ctsb and Ctsz genotypes as PymT+/0;wt, PymT+/0;Ctsb−/−, PymT+/0;Ctsz−/−, and PymT+/0;Ctsb−/−Ctsz−/−, and the four respective cathepsin genotypes without the PymT transgene. Ctsz−/− mice show no gross phenotype for observation periods of up to 2 years and can be maintained as homozygous mutants. In addition, we investigated the postnatal development of Ctsb−/−Ctsz−/− mice with respect to weight gain and steroid hormone levels. There was no evidence of impaired quality of maternal care or overall health of the Ctsb−/−Ctsz−/− mice (Fig. S2A). The offspring of wt and Ctsb−/−Ctsz−/− weighed the same at weaning on day 21 after birth and demonstrated equal weight gain (Fig. S2B). Quantification of steroid hormone concentrations in serum of wt and Ctsb−/−Ctsz−/− mice at day 42 after birth by ELISA revealed no differences in circulating levels of estrogen and progesterone, indicating a similar onset of puberty (Fig. S2C). Most importantly, similar levels of steroid hormones suggest comparable activation of the MMTV promoter, which is costimulated by steroid hormones. To further address PymT oncogene expression, we measured the PymT mRNA levels in tumors of 14-week-old PymT+/0;wt, PymT+/0;Ctsb−/−, PymT+/0;Ctsz−/−, and PymT+/0;Ctsb−/−Ctsz−/− mice by quantitative RT-PCR (Fig. S2D). These experiments revealed no cathepsin-genotype dependent differences in PymT expression. Thus, any alteration of tumor progression in the cathepsin-deficient MMTV-PymT mice is not caused by impaired postnatal development or reduced oncogene levels, but rather is most likely a consequence of altered cathepsin expression in the various knockout mice. Expression Pattern, Activity Profile, and Histological Localization of Ctsb and Ctsz. Western blot analyses of Ctsb and Ctsz (Fig. 1A)
revealed the expected expression pattern in the corresponding groups, with PymT+/0;wt expressing both Ctsb and Ctsz, PymT+/0; Ctsb−/− lacking expression of Ctsb, and PymT+/0;Ctsz−/− lacking
Fig. 1. Expression pattern of Ctsb and Ctsz in PymT-induced mammary carcinomas. (A and B) Detection of Ctsb and Ctsz expression by Western blot analyses in primary tumor lysats (A) and cell surface labeling of active cysteine proteases by the biotinylated activity–based probe DCG-04 (B) on primary tumor cells of PymT+/0;wt [1], PymT+/0;Ctsb−/− [2], PymT+/0;Ctsz−/− [3], and PymT+/0;Ctsb−/−Ctsz−/− [4] mice. Unlabeled PymT+/0;wt control [5] was analyzed to detect endogenously biotinylated proteins. α-tubulin served and colloidal Coomassie blue–stained gel served as loading controls. (C) Detection of Ctsz (green staining) by immunofluorescence staining of PymT+/0;wt, PymT+/0;Ctsb−/−, and PymT+/0;Ctsz−/− tumor sections. (Scale bar: 50 μm.)
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expression of Ctsz. PymT+/0;Ctsb−/−Ctsz−/− showed no expression of Ctsb and Ctsz. Having recently reported the extracellular expression of Ctsz as a second cysteine cathepsin on PymT tumor cells (20), we analyzed activity profiles of cysteine cathepsins on the surface of PymT tumor cells with different Ctsb and Ctsz genotypes (Fig. 1B). Cell surface labeling of cysteine cathepsins with the membrane-impermeable active site probe DCG-04 (29) revealed the presence of Ctsb and Ctsz on PymT+/0;wt cells. PymT+/0;Ctsb−/− and PymT+/0;Ctsz−/− showed distinct cysteine cathepsin bands considered to be Ctsz on Ctsb-deficient tumor cells and Ctsb on Ctsz-deficient tumor cells. Notably, no active cysteine cathepsins were detectable on the surface of PymT+/0; Ctsb−/−Ctsz−/− tumor cells. Because Ctsz has not yet been investigated in the context of PymT-induced mammary cancer, we analyzed the histological localization of Ctsz in primary PymT tumors of PymT+/0;wt, PymT+/0;Ctsb−/−, and PymT+/0;Ctsz−/− as control (Fig. 1C). Ctsz expression was detected in tumor cells and cells of the tumor stroma. Interestingly, immunofluorescence staining of Ctsz revealed particularly high Ctsz expression in PymT+/0;Ctsb−/− tumors. Moreover, Ctsb and Ctsz showed the same expression pattern in primary PymT tumors and lung metastases (SI Methods and Fig S3). Progression and Histopathology of PymT-Induced Mammary Carcinomas. To detect first palpable tumors, we palpated 10 mam-
mary glands per mouse every second day in a genotype-blinded fashion starting on day 28 after birth. To assess genotype-dependent differences in the occurrence of first palpable tumors, we determined the time point at which each genotype developed 50% of the tumors (Fig. 2A). The PymT+/0;Ctsb−/− and PymT+/0;Ctsz−/− mice had an average of five palpable tumors 4.5 days later than the PymT+/0;wt mice (P
