Involvement of the lysosomal cysteine peptidase cathepsin B in tumour progression and formation of metastases

The leading cause of cancer deaths is tumour metastasis. The formation of metastases is a complex multistep process, involving invasion of the basal membranes and connective tissue, penetration of the blood vessel walls, invasion of the host tissue, and initiation of growth at the new location. The degradation of extracellular matrix and basal membranes by proteolytic enzymes makes possible the invasive growth of tumour cells. Numerous reports describe increased levels of proteolytic enzymes in invasive tumours. These proteases include neutral metalloproteinases, serine proteinases as well as lysosomal, aspartic and cysteine peptidases. <br>To investigate the role of the lysosomal cysteine peptidase cathepsin B (CTSB) in tumour progression and metastasis, transgenic mice FVB/N-TgN(MMTVPyVT)634Mul, developing multifocal metastasising mammary carcinomas, were utilised. CTSB deficient mice, with a mixed C57BL/6J and 129SvJ background were made congenic for the FVB/N strain. FVB/N-TgN(MMTVPyVT)-CTSB, wt, ht and ko were generated through inter-crossing (PyCB strain). The mice were sacrificed sixty days after the discovery of the first palpable mammary tumour. The weight of primary tumours was measured and the volume of the pulmonary metastases was evaluated by histomorphometry. <br>The genotype was found to be an influential factor on the primary tumour weight as well as on the total metastasis volume in the lungs but played no role on the timing of the initiation of the primary mammary tumour. Statistical analysis showed that the adjusted mean of primary tumour weight for the Py;Ctsb+/+ (n=36) is significantly higher compared to Py;Ctsb+/-(n=38, p=0.006) and Py;Ctsb-/- (n=32, p=0.001). Surprisingly, in the case of the total lung metastasis volume, the adjusted means of the genotypes Py;Ctsb+/+ and Py;Ctsb-/- are not significantly different (p=0.13) but there is a significant difference between Py; Ctsb+/+ andPy; Ctsb+/- (p=0.01). <br>Histological evaluation of the PyCB female mice, of the three different genotypes, shows no difference in tumour development and extravasation as well as in lung metastasis development. <br>In order to study the formation of colonisation and metastasis, independently from the primary tumour formation, 5 x 105 primary tumour cells of different CTSB genotypes were i.v. injected into FVB/N congenic females of different CTSB genotypes. The animals were sacrificed two weeks after the injection and the lung metastases were evaluated. The total volumes of the metastases were significantly smaller, when ctsb+/+ tumour cells were injected into ctsb-/- females, compared to ctsb+/- or ctsb+/+ recipients (ctsb-/- 0.92 mm3 (&#61617; 0.42), ctsb+/- 2.37 mm3 (&#61617; 1.09), p= 0.0013; ctsb+/+ 2.16 mm3 (&#61617; 0.99), p=0.006). No differences in the total metastasis volume were observed with the injection of ctsb-/- tumour cells, compared to ctsb+/+. <br>In situ hybridisation of mammary tumour tissue and lung metastatic tissue of FVB/N-TgN(MMTVPyVT)634Mul female mice showed a high expression of cathepsin B and cathepsin L in the area of the tumour. In the case of the lung metastases the expression of both cathepsins was concentrated on the borders of the metastasis suggesting that the cells that surround the metastasis might produce CTSB and CTSL. Furthermore, immunohistological analysis of the lung metastatic tissue showed that most of these cells were macrophages, as staining with the macrophage specific antibody F4/80 confirmed. Double immunofluorescence revealed that these tumour-surrounding macrophages express high levels of CTSB. <br>These data indicate that extracellular CTSB produced by inflammatory stromal macrophages plays a role in primary tumour growth as well as in metastasis formation and growth. CTSB expression enhances tumour growth. However, Py;ctsb+/- and Py;ctsb-/- tumour cells proliferate less compared to Py;ctsb+/+ but Py;ctsb-/- tumour cells have a higher ability to degrade extracellular matrix proteins than Py;ctsb+/+ and Py;ctsb+/- tumour cells.

Author dc:creator

• Papazoglou, Anna

Contributors dc:contributor

• Peters, Christoph

• Breast cancer
• Lung metastasis
• Cathepsin B

Repository record source_url

https://freidok.uni-freiburg.de/data/1143

OAI identifier oai:identifier

oai:freidok.uni-freiburg.de:1143