Cathepsin D–many functions of one aspartic protease. Crit Rev Oncol Hematol. extent than after L1-transfection. The suppression of endogenous CTSD in L1-expressing cells blocked the increase in the proliferative, motile, tumorigenic and metastatic ability of CRC cells. Enhancing Wnt/-catenin signaling by the inhibition of GSK3 resulted in increased endogenous CTSD levels, suggesting the involvement of the Wnt/-catenin pathway in CTSD expression. In human CRC tissue, CTSD was detected in Saquinavir Mesylate epithelial cells and in the stromal compartment at the more invasive areas of the tumor, but not in the normal mucosa, indicating that CTSD plays an essential role in CRC progression. test. When analyzing the effects of changes in CTSD levels in CRC cells lacking or expressing L1, we observed a similar effect on cell motility (by the scratch wound closure experiment) and tumor growth in mice upon s.c injection (Figure 2DC2G). Thus, CTSD overexpression resulted in a modest, yet significant, increase in LS 174T cell motility (Figure 2D) and the suppression of endogenous CTSD levels in CRC cells stably expressing L1, reduced their motility (Figure 2E). The injection of these CRC cell clones s.c into immunocompromised mice resulted in a small increase in tumor formation upon CTSD overexpression (Figure 2F, compare CTSD cl 1 and 2 to L1), while CTSD suppression in L1 expressing cells resulted in a marked reduction in tumorigenic capacity of these cells (Figure 2F and ?and2G,2G, compare L1+shCTSD cl1 and cl2 to L1). We have also studied the possible effects of CTSD on the ability of L1 to confer liver metastasis upon injecting the cells into the spleen  and following the formation of metastases in the liver. CRC cell clones stably overexpressing L1 very effectively formed liver metastases upon their injection into the spleen of mice (Figure 3B compare to 3A and ). The overexpression of CTSD alone also induced liver metastasis (Figure 3C), but to a lesser extent than L1 overexpression (compare Figure 3C to 3B, Supplementary Figure 2). CRC cells overexpressing L1 in which the endogenous CTSD levels were suppressed, had a dramatically reduced capacity to form metastases in the liver (Figure 3D), although they continued expressing L1 (Supplementary Figure 2). Taken together, the results described in Figures 2 and ?and33 demonstrated that while CTSD can promote the motile and tumorigenic capacity of CRC cells, CTSD is much less potent than L1 in conferring tumorigenic properties. On the other hand, in the context of L1-mediated effects on the tumorigenic and metastatic capacities of CRC cells, the increase in CTSD expression is essential for the L1-conferred tumorigenic properties. Open in a separate window Figure 3 CTSD expression levels affect the metastatic ability of human CRC Saquinavir Mesylate cells to the liver.The ability of the LS 174T cell clones described in (Figure 2A) to form liver metastases was determined by injecting 2 106 cells into the spleen of nude mice for each cell line and excising the liver and spleen of such mice after 6 weeks. In control pcDNA3-transfected (A) and L1-transfected cells (B) the results with only two mice are shown. (C) CTSD overexpressing LS 174T cell clones (CTSD cl1 and cl2), and (D) L1+shCTSD cell clones (cl1 and cl2). The white areas in the liver tissue represent the metastatic lesions formed by the human CRC cells. The white arrows in (D) point to the much smaller metastatic foci formed when the levels of CTSD were suppressed in L1-expressing cells with shRNA to CTSD. The increase in CTSD by L1 is mediated by enhanced Wnt/-catenin signaling In previous studies we have shown that L1 exerts its downstream effects by signaling through the NF-B pathway [12, 19]. We have therefore analyzed the levels of CTSD in L1-overexpressing CRC cell clones in which the Tead4 signaling by NF-B was blocked, either by expressing the IB super repressor (IB-SR), or by reducing the level of the p65 NF-B subunit using shRNA to p65 (Figure 4A). The inhibition of NF-B signaling by these methods had no effect on the induction of CTSD in L1-overexpressing CRC cells (Figure 4A), suggesting that L1 induces CTSD via different signaling pathways. Open in a separate window Figure 4 Regulation of CTSD expression by L1 does not involve NF-B but is affected by Wnt/-catenin signaling.(A) NF-B signaling was blocked in L1-expressing CRC cell clones by stably expressing the mutant IB super repressor IB-SR (L1+IB-SR cl1 and cl2), or by suppressing the NF-B subunit p65 Saquinavir Mesylate using shp65RNA (L1+shp65 cl1 and cl2)..