02 BOOK OF ABSTRACTS .indd

Involvement of the lysosmal cysteine peptidase Cathepsin
L in progression of epidermal carcinomas
Tobias Lohmüller, Julia Dennemärker, Ulrike Reif, Susanne Dollwet-Mack,
Christoph Peters and Thomas Reinheckel
Department of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg,
Germany
Papain-like cysteine proteases have been implicated to work as effectors of
invasive growth and neovascularisation. In order to assess the functional
significance of cathepsin L (CTSL) during neoplastic progression in a transgenic
mice model of multistage epidermal carcinogenesis, we adopted a
genetic approach utilizing cathepsin L-knockout mice breed with transgenic
Tg(K14-HPV16) mice, which express the human papillomavirus type 16 oncogenes
under the control of the human keratin 14 promoter, and reproducibly show multistage
development of invasive squamous cell carcinomas of the epidermis [1]. Before the
intercross of the two mouse lines, CTSL-deficient mice have been backcrossed to
the FVB/n genetic background for 8 generations to ensure a congenic tumor model
Tg(K14-HPV16);ctsl -/- . To analyse cathepsin expression in the tumor model we quantified
mRNA expression mRNA expression of cathepsins B, L, H, D, and X at various stages
of tumorigenesis by real time PCR. While CTSL was absent in Tg(K14-HPV16);ctsl -/-
mice, no other genotype specific differences could be observed for comparison of
Tg(K14-HPV16);ctsl +/+ and Tg(K14-HPV16);ctsl -/- mice. Immunohistochemistry (IHC)
revealed that CTSL lost its strong suprabasal epidermal staining during carcinogenesis
was uniformly distributed in cancers, while CTSB maintained its staining pattern.
Interestingly, IHC quantification of the proliferation marker Ki67 showed significant
higher proliferation rates in epidermis of 16 and 24 weeks old Tg(K14-HPV16);ctsl -/-
mice. Further, the progression from hyperplasia to dysplasia occurs significantly faster
in tumor mice with CTSL-deficiency. However, no general differences in keratinocyte
differentiation, as assessed by cytokeratin 5 and 10 IHC, and in angiogenesis, as
quantified by CD31-IHC and FACS analyses, could be detected. Compared to
K14-HPV16 mice that are heterozygous or wild-type for CTSL, analysis of
CTSL-deficient tumor mice demonstrated significant differences in squamous cell
carcinoma onset and development to a tumor volume of 1cm 3 . The mean onset of
the first palpable tumors in ctsl -/- , ctsl +/- and ctsl +/+ K14-HPV16 animals were at 28, 36
and 37 weeks of age, and the mean age for occurrence of a 1cm 3 tumor was at 32, 43
and 42 weeks, respectively. In addition, tumors of CTSL knockout mice developed
significantly higher grade (i.e. less differentiated) carcinomas than wild-type and
heterozygous controls. Further, the number of lymph node metastases significantly
increased in Tg(K14-HPV16);ctsl -/- mice.
Taken together, CTSL deficiency promotes multistage carcinogenesis in K14 HPV16
mice, results in highly dedifferentiated cancers and an increased metastatic capacity.
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[1] Coussens, L.M., D. Hanahan, and J.M. Arbeit. 1996. Genetic predisposition and
parameters of malignant progression in K14-HPV16 transgenic mice. Am J Pathol.
149:1899-917.