Increased expression of cathepsin D is required for L1-mediated colon cancer progression

Abstract

Hyperactivation of Wnt/β-catenin target genes is considered a key step in human colorectal cancer (CRC) development. We previously identified the immunoglobulin-like cell adhesion receptor L1 as a target gene of β-catenin/TCF transactivation that is localized at the invasive edge of CRC tissue. Using gene arrays, we discovered a number of downstream target genes and signaling pathways conferred by L1 overexpression during colon cancer progression. Here, we have used a proteomic approach to identify proteins in the secretome of L1-overexpressing CRC cells and studied the role of the increase in the aspartate protease cathepsin D (CTSD) in L1-mediated colon cancer development. We found that in addition to the increase in CTSD in the secretome, the RNA and protein levels of CTSD were also induced by L1 in CRC cells. CTSD overexpression resulted in elevated proliferation under stress and increased motility, tumorigenesis and liver metastasis, although to a lesser 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/β-catenin signaling by the inhibition of GSK3β resulted in increased endogenous CTSD levels, suggesting the involvement of the Wnt/β-catenin pathway in CTSD expression. In human CRC tissue, CTSD was detected in 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.

Keywords: L1; Wnt/β-catenin signaling; cathepsin D; colon cancer; metastasis.

Figure 1. L1 overexpression in human CRC cell lines induces the expression of CTSD.

(A) The levels of CTSD RNA (determined by RT-PCR) in L1-expressing LS 174T CRC cell clones are higher than in control pcDNA3-transfected cells. (B) Western blotting showing that CTSD protein levels are higher in L1-expressing CRC cells compared to control (pcDNA3 transfected cells) and (C), secreted CTSD levels in the culture medium are higher in L1-expressing LS 174T CRC cells. The same findings were seen in the human HCT 116 CRC cell line transfected with L1 that showed increased, (D) CTSD RNA, (E) CTSD protein and (F) CTSD protein secreted into the culture medium. (G) Endogenous CTSD immuno-staining (red) was much stronger in L1-transfected LS 174T cells (green), compared to control LS 174T cells (pcDNA3). Nuclei were stained with DAPI (blue). The bar represents 20 μm. Ponceau staining of the western blots served for determining equal loading of the gels.

Figure 2. Modulating the expression of CTSD in CRC cells affects cell growth, motility and tumorigenesis.

(A) Clones of LS 174T cells stably transfected with CTSD (CTSD cl1 and cl2), control pcDNA3 and L1-transfected LS 174T cells in which endogenous levels of CTSD were suppressed by shRNA (L1+shCTSD cl1 and cl2) were isolated. The expression of the relevant proteins was determined by western immunoblotting. (B) The proliferation in 0.1% serum of CTSD-overexpressing cells was compared to that of cells transfected with L1, or with pcDNA3. (C) The proliferation of LS 174T expressing L1+shCTSD cell clones was compared to that of cells expressing L1, or pcDNA3, over 5 days. (D, E) The motility of the LS 174T cell clones described in (A) was determined by the “scratch wound” experiment 24 hours after introducing the artificial wound in triplicate samples for each cell clone. (F, G) The tumorigenic capacity of the cell clones described in (A) was determined 2 weeks after s.c injection into nude mice of 10⁶ cells for each clone. (F) The tumors were excised and photographed and their weight (G) was determined. ^* P < 0.05. Two-sided Student’s t test.

Figure 3. CTSD expression levels affect the metastatic ability of human CRC 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.

Figure 4. Regulation of CTSD expression by L1 does not involve NF-κB but is affected by Wnt/β-catenin signaling.

(A) NF-κB signaling was blocked in L1-expressing CRC cell clones by stably expressing the mutant IκBα super repressor IκB-SR (L1+IκB-SR cl1 and cl2), or by suppressing the NF-κB subunit p65 using shp65RNA (L1+shp65 cl1 and cl2). The levels of endogenous CTSD and NF-κB signaling proteins were determined in these cell clones by western blotting with the appropriate antibodies. (B) Treatment with 30 mM LiCl was used to enhance β-catenin-TCF transactivation (treatment with NaCl served as control) and the level of CTSD was determined in pcDNA3 and L1-expressing LS 174T cell clones. The levels of phosphorylated GSK-3, total β-catenin and total GSK-3 were determined. Tubulin and Ponceau staining served as controls for equal loading of the gels. Note that treatment with L1Cl resulted in the elevation of the inactive p-GSK3α and β and increased CTSD levels, both in pcDNA3 and L1-expressing cells, suggesting that β-catenin-TCF signaling is involved in CTSD expression. No changes in total β-catenin and GSK-3 levels were detected. Inhibition of NF-κB signaling had no effect on CTSD levels in L1-expressing cells.

Figure 5. CTSD is expressed in human CRC tissue and in invasive areas of the stroma.

Formaldehyde-fixed and paraffin embedded human CRC samples were analyzed by immunohistochemistry for CTSD presence. (Α) In 30 out of 38 samples the CRC tissue samples displayed strong CTSD staining of the epithelial tissue (black arrows) and, in addition, all CRC tissue samples were positive for CTSD in the stroma (red arrow). (B) The normal colon mucosa was not stained with anti CTSD antibody. (C) The invasive stromal area (blue line) was often strongly stained for CTSD (red arrows). The bar in (C) represents 300 μm.