Thrombin up-regulates cathepsin D which enhances angiogenesis, growth, and metastasis.

Cathepsin D (CD) up-regulation has been associated with human malignancy and poor prognosis. Thrombin up-regulated CD mRNA and protein in eight tumor cell lines as well as in human umbilical vascular endothelial cells (HUVEC). Thrombin increased the secretion of CD by 3- to 8-fold and enhanced chemotaxis ( approximately 2-fold) in 4T1 murine mammary CA cells, which was completely inhibited with the knockdown of CD. Secreted 4T1 CD induced neoangiogenesis by 2.4-fold on a chick chorioallantoic membrane, which was blocked in CD-KD cells. The addition of pure CD (2 ng) to the chick chorioallantoic membrane increased angiogenesis by 2.1-fold, which was completely inhibited by Pepstatin A (Pep A). CD enhanced human HUVEC chemotaxis and Matrigel tube formation by 2-fold, which was then blocked by Pep A. CD enhanced HUVEC matrix metalloproteinase 9 (MMP-9) activity by approximately 2-fold, which was completely inhibited by Pep A as well as a generic MMP inhibitor, GM6001. The injection of CD-KD 4T1 cells into syngeneic mice inhibited tumor growth by 3- to 4-fold compared with empty vector (EV) cells. Hirudin, a specific thrombin inhibitor, inhibited the growth of wild-type and EV cells by 2- to 3-fold, compatible with thrombin up-regulation of CD. CD and thrombin also contributed to spontaneous pulmonary metastasis; 4-fold nodule inhibition with CD versus EV and 4.6-fold inhibition with hirudin versus EV (P < 0.02). Thus, thrombin-induced CD contributes to the malignant phenotype by inducing tumor cell migration, nodule growth, metastasis, and angiogenesis. CD-induced angiogenesis requires the proteolytic activation of MMP-9.