Wnt9A Induction Linked to Suppression of Human Colorectal Cancer Cell Proliferation

Abstract

Most studies of Wnt signaling in malignant tissues have focused on the canonical Wnt pathway (CWP) due to its role in stimulating cellular proliferation. The role of the non-canonical Wnt pathway (NCWP) in tissues with dysregulated Wnt signaling is not fully understood. Understanding NCWP's role is important since these opposing pathways act in concert to maintain homeostasis in healthy tissues. Our preliminary studies demonstrated that LiCl inhibited proliferation of primary cells derived from colorectal cancer (CRC). Since LiCl stimulates cell proliferation in normal tissues and NCWP suppresses it, the present study was designed to investigate the impact of NCWP components in LiCl-mediated effects. LiCl-mediated inhibition of CRC cell proliferation (p < 0.001) and increased apoptosis (p < 0.01) coincided with 23-fold increase (p < 0.025) in the expression of the NCWP ligand, Wnt9A. LiCl also suppressed β-catenin mRNA (p < 0.03), total β-catenin protein (p < 0.025) and the active form of β-catenin. LiCl-mediated inhibition of CRC cell proliferation was partially reversed by IWP-2, and Wnt9A antibody. Recombinant Wnt9A protein emulated LiCl effects by suppressing β-catenin protein (p < 0.001), inhibiting proliferation (p < 0.001) and increasing apoptosis (p < 0.03). This is the first study to demonstrate induction of a NCWP ligand, Wnt9A as part of a mechanism for LiCl-mediated suppression of CRC cell proliferation.

Keywords: LiCl; Wnt pathway ligand; Wnt9A; canonical Wnt pathway; human colorectal cancer; non-canonical Wnt pathway; β-catenin.

Figure 1

Effect of LiCl on proliferation and apoptosis of colorectal cancer (CRC) cells. CRC cell proliferation and apoptosis was determined in cells treated with LiCl for a period of 72 h. (A) Proliferation determined in cells treated with different concentrations of LiCl (n = 4); (B) Proliferation determined in cells treated with 20 mM LiCl (n = 5); (C) Apoptosis determined in cells treated with 20 mM LiCl (n = 5). Statistical analysis: One way ANOVA with post hoc pairwise multiple comparisons using Student-Newman-Keuls method and Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

The effect of LiCl on β-catenin message and protein in CRC cells. CRC cells were treated for various periods of times with 20 mM LiCl and β-catenin mRNA and protein were determined. (A) β-catenin mRNA in cells treated with LiCl for 72 h (n = 4); (B) Total β-catenin protein in cells treated for 24, 48 and 72 h (n = 5); (C) Active β-catenin protein in cells treated with LiCl for 24, 48 and 72 h (n = 2); Statistical analysis: (D) Active β-catenin protein in cells treated with LiCl for 72 h (n = 4). One way ANOVA with post hoc pairwise multiple comparisons using Student-Newman-Keuls method and Student’s t-test. ^α p < 0.03; * p < 0.025; ** p < 0.01; *** p < 0.001.

Figure 3

The effect of LiCl on the expression of Wnt signaling components. CRC cells were treated with 20 mM LiCl for a period of 72 h and gene expression analysis was performed. (A) Heat map showing the intensity of Wnt pathway gene expression changes induced by LiCl in all five CRC cells. Data shown is of gene expression changes of two-folds or greater in four or all five cell lines; (B) Summary of the LiCl-mediated changes in gene expression from the heat map in panel A. The data are expressed as means ± SEM. of fold change relative to media control from 5 distinct CRC cell lines. Statistical analysis: Student’s t-test. * p < 0.025; ** p < 0.01; n = 5. The intensity of gene expression increases from white to black with the black color representing genes expressed the greatest.

Figure 4

Effect of IWP-2 and Wnt9A antibody on LiCl-mediated suppression of CRC cell proliferation. (A) CRC cells were treated with 20 mM LiCl with or without 1.0 µM IWP-2 (n = 4); (B) Effect of conditioned media from cells treated with media, LiCl, IWP-2 or LiCl+IWP-2 on CRC proliferation (n = 4); (C) Cell proliferation was determined in CRC treated with LiCl, LiCl + IgG, or LiCl + Wnt9A antibody (n = 5). The data are expressed as means ± SEM. of fold change relative to media or LiCl controls. Statistical analysis: One way ANOVA with post hoc pairwise multiple comparisons using Student-Newman-Keuls method. * p < 0.05; ** p < 0.01; *** p < 0.005. NS = not statistically significant.

Figure 5

Effect of recombinant Wnt9A protein on CRC cell proliferation, apoptosis and active β-catenin levels. CRC cells were treated with recombinant human Wnt9A protein for a period of 72 h. (A) Proliferation determined in cells treated with a range of Wnt9A protein; (B) Apoptosis was determined in cells treated with 500 ng/mL Wnt9A protein; (C) Active β-catenin protein in cells treated with 500 ng/mL Wnt9A protein. Statistical analysis: Student’s t-test. * p < 0.03; ** p < 0.01; *** p < 0.001; n = 5.

Figure 6

Mechanism for the LiCl-mediated suppression of CRC cell proliferation. Schematic shows the mechanism involved in the LiCl-mediated suppression of CRC cell proliferation. Blunted arrows indicate inhibition. Dotted line indicates unknown mechanism.