The Notch-2 gene is regulated by Wnt signaling in cultured colorectal cancer cells

Abstract

Background: Notch and Wnt pathways are key regulators of intestinal homeostasis and alterations in these pathways may lead to the development of colorectal cancer (CRC). In CRC the Apc/β-catenin genes in the Wnt signaling pathway are frequently mutated and active Notch signaling contributes to tumorigenesis by keeping the epithelial cells in a proliferative state. These pathways are simultaneously active in proliferative adenoma cells and a crosstalk between them has previously been suggested in normal development as well as in cancer.

Principal findings: In this study, in silico analysis of putative promoters involved in transcriptional regulation of genes coding for proteins in the Notch signaling pathway revealed several putative LEF-1/TCF sites as potential targets for β-catenin and canonical Wnt signaling. Further results from competitive electrophoretic mobility-shift assay (EMSA) studies suggest binding of several putative sites in Notch pathway gene promoters to in vitro translated β-catenin/Lef-1. Wild type (wt)-Apc negatively regulates β-catenin. By induction of wt-Apc or β-catenin silencing in HT29 cells, we observed that several genes in the Notch pathway, including Notch-2, were downregulated. Finally, active Notch signaling was verified in the Apc(Min/+) mouse model where Hes-1 mRNA levels were found significantly upregulated in intestinal tumors compared to normal intestinal mucosa. Luciferase assays showed an increased activity for the core and proximal Notch-2 promoter upon co-transfection of HCT116 cells with high expression recombinant Tcf-4, Lef-1 or β-catenin.

Conclusions: In this paper, we identified Notch-2 as a novel target for β-catenin-dependent Wnt signaling. Furthermore our data supports the notion that additional genes in the Notch pathway might be transcriptionally regulated by Wnt signaling in colorectal cancer.

Figure 1. In silico analysis of Notch pathway gene promoters.

In silico analysis of the genetic networks directly involved in Notch and Wnt signaling suggest overlap and direct crosstalk via Notch target gene activation through canonical Wnt signaling. By means of MatInspector, gene promoters in the Notch pathway were found to contain at least one putative LEF-1/TCF-site (number of sites in per gene is described in square brackets). Genes in gray boxes were subjected for further semi quantitative RT-PCR analysis.

Figure 2. In vitro translated β-catenin/Lef-1 binds to Notch pathway gene promoters.

Competitive electro mobility-shift assay of the proximal Notch-2 promoter. Duplex CD1TOP probes were “end labeled” with [³²P]dATP, incubated with in vitro translated β-catenin/Lef-1 and exposed to competition with abundance of cold or cold mutated promoter duplex oligonucleotides (×300 and ×900, respectively). The protein-DNA complexes were separated by electrophoresis and visualized by autoradiography. As a competition control cold CD1TOP and cold mutated CD1FOP competed with radiolabeled CD1TOP and to confirm that radioactive labeled CD1TOP binds Lef-1 specifically, plasmid-free reticulocyte lysate were subjected to in vitro translation and incubated with radiolabeled probe. Gene numbering describe position of the LEF-1/TCF-site relative the gene translation start site. Adjustments in whole image contrast levels were performed in Adobe Photoshop CS4.

Figure 3. The expression of Notch pathway genes are dependent of Apc status in HT29 cells and murine intestinal adenomas.

Wt-Apc was induced in HT29-APC cells through the addition of 100 µM ZnCl₂ to the growth medium. Semi-quantitative RT-PCR was carried out on cDNA reversely transcribed from 200 ng total RNA from each time point (0–24 h post wt-Apc induction). Bars describe the relative expression of Notch-2 in HT29-APC cells (*) versus HT29-β-galactosidase cells (**) normalized against Gapdh expression. Cyclin D1 was used as a positive control for Wnt inactivation. (B) The protein expression of Apc (∼310 kDa), Notch-2 (∼265 kDa), wt and loading control, Gapdh, was determined with Western blot following 0–24 h of zinc stimulation. Adjustments in whole image contrast levels were performed in Adobe Photoshop CS4. (C) Relative mRNA expression of murine Notch-1, Notch-2 and Hes-1 in tumors and corresponding non-tumor normal intestinal mucosa of Apc^Min/+ mice. mRNA expression was related to the endogenous control gene GAPDH. White columns (n = 16), black columns (n = 22). Bars are presented as median expression values. Error bars describe interquartile range.

Figure 4. The Notch-2 promoter contains four putative LEF1/TCF-sites and results in high luciferase gene activity.

(A) Schematic representation of the proximal Notch-2 promoter with the four putative LEF-1/TCF-binding consensus sites, identified with Genomatix MatInspector, at positions −2261, −869, .689 and −110 relative translational start site (ATG). A potential transcription start site was mapped to position −256 using Genomatix Gene2Promoter and PromoterInspector software. Uppercase letters indicate the core consensus sequence. (B) N2PR −2327/−99, N2PR −110 in pGL3-TATA as well as empty pGL3-TATA (Smith et al, 2002 [40]) were transfected into HCT116 and and co-transfected with pSV-β-galactosidase control vector. The cell lysate 24 h post transfection was subjected to luciferase reporter assays and relative luciferase activity determined. Error bars describe SEM.

Figure 5. The Notch-2 promoter is transcriptionally activated via Lef-1/Tcf-4/ß-catenin mediated signaling.

(A) HCT116 cells were co-transfected with pGL3-TATA carrying N2PR −2327/−99 (or empty pGL3-TATA) and HA-S33Y-β-catenin in pCGN, hTcf-4 or mLef-1 in pcDNA6 as well as pSV-β-galactosidase control vector (n = 6). (B) HCT116 cells were co-transfected with pGL3-TATA carrying N2PR −110 (white columns) or mutated N2PR −110 SpeI (black columns) (or empty pGL3-TATA) and HA-S33Y-β-catenin in pCGN, hTcf-4 or mLef-1 in pcDNA6 as well as pSV-β-galactosidase control vector. Luciferase activity was normalized against pGL3-TATA background activity by dividing N2PR −2327/−99-pGL3-TATA relative expression with the relative expression from empty luciferase vector (n = 9). Error bars describe SEM. (C) ChIP in HCT116 cells co-transfected with His-tagged Lef-1 or Tcf-4, N2PR −2327/−99 and N2PR −110; immunoprecipitation with anti-His (6× His tag® antibody) or IgG control, PCR with primers encompassing LEF-1/TCF-site −110 in the Notch-2 promoter. A 172-bp segment of NCAPD2 promoter was used as a negative control. (D) Western blots with anti-His (6× His tag® antibody) verifying the transfection of His-tagged Lef-1 (∼65 kDa) and Tcf-4 (∼45 kDa) in HCT116. Adjustments in whole image contrast levels were performed in Adobe Photoshop CS4.