Frizzled-7 as a potential therapeutic target in colorectal cancer

Abstract

We investigated whether one of the Wnt receptors, frizzled-7 (FZD7), functions in the canonical Wnt signaling pathway of colorectal cancer (CRC) cells harboring an APC or CTNNB1 mutation and may be a potential therapeutic target for sporadic CRCs. The expression level of FZD gene family members in colon cancer cells and primary CRC tissues were determined by real-time PCR. Activation of the Wnt signaling pathway was evaluated by TOPflash assay. The expression level of Wnt target genes was determined by real-time polymerase chain reaction and/or Western blot analysis. Cell growth and cell invasion were assessed by MTS and matrigel assays, respectively. Among 10 FZD gene family members, FZD7 mRNA was predominantly expressed in six colon cancer cell lines with APC or CTNNB1 mutation. These six cell lines were transfected with FZD7 cDNA together with a TOPflash reporter plasmid, resulting in a 1.5- to 24.3-fold increase of Tcf transcriptional activity. The mRNA expression levels of seven known Wnt target genes were also increased by 1.5- to 3.4-fold after transfection of FZD7 cDNA into HCT-116 cells. The six cell lines were then cotransfected with FZD7-siRNA and a TOPflash reporter plasmid, which reduced Tcf transcriptional activity to 20% to 80%. FZD7-siRNA was shown to significantly decrease cell viability and in vitro invasion activity after transfection into HCT-116 cells. Our present data demonstrated that FZD7 activates the canonical Wnt pathway in colon cancer cells despite the presence of APC or CTNNB1 mutation and that FZD7-siRNA may be used as a therapeutic reagent for CRCs.

Figure 1

The mRNA and protein expression level of frizzled family members in colon cancer cell lines. (A) The extracted total RNA was reverse-transcribed into single-stranded cDNA. Real-time PCR was performed using first-strand cDNA with TaqMan. GAPDH was used as an endogenous control. Data are presented as mean values and SD for three independent experiments compared with the level of FZD1 that is presented as 1. (B) Western blot analysis of protein expression of FZD7 in six colon cancer cells. 293T cells was transfected with FZD7 as a positive control.

Figure 2

Effects of FZD7 overexpression on canonical Wnt signaling. (A) Effects of FZD7 overexpression on Tcf-reporter transcriptional activiry. Colon cancer cell lines were transiently cotransfected with either mock, FZD1, FZD6, or FZD7, TOPflash reporter plasmid, and pRL-TK plasmid encoding Renilla luciferase as an internal control for transfection efficiency. At 48 hours after transfection, cell lysates were measured for relative luciferase activities. Data are presented as mean values and SD for three independent experiments compared with the level of luciferase activity obtained in the presence of mock that is presented as 1. (B) Real-time PCR analysis of expression of Wnt target genes in HCT-116 cells transfected with mock, FZD1, or FZD7. HCT-116 cells were transiently transfected with mock, FZD1, or FZD7. At 48 hours after transfection, total RNA were reverse-transcribed and amplified with primers specific for each indicated Wnt target as described in the Materials and Methods section. (C) Western blot analysis of expression of Wnt target genes in HCT-116 cells transfected with mock, FZD1, or FZD7. HCT-116 cells were transiently transfected with mock, FZD1, or FZD7. At 48 hours after transfection, each cytosolic protein was prepared, and 10 µg of each was subjected to electrophoresis. Expression of β-catenin, Myc, Survivin, and Jun were assessed. β-Actin served as a loading control.

Figure 3

Effects of FZD7-siRNA on canonical Wnt signaling. (A) FZD7-siRNA specifically inhibits the expression of FZD7. 293T cells were transiently transfected with various combinations of pEF4, pEF4-FZD-V5, piGENE, and FZD7-siRNA. A total of 30 µg of cell lysates was analyzed by Western blot analysis using anti-V5 or anti-β-actin antibodies. (B) Effects of FZD7-siRNA on Tcf-reporter transcriptional activity. Colon cancer cell lines were transiently cotransfected with either piGENE or FZD7-siRNA, TOPflash reporter vector, and pRL-TK plasmid encoding Renilla luciferase as an internal control for transfection efficiency. At 48 hours after transfection, cell lysates were measured for relative luciferase activities. Data are presented as mean values and SD for three independent experiments compared with the level of luciferase activity obtained in the presence of piGENE that is presented as 1. (C) Western blot analysis of the expression of Wnt target genes in HCT-116 cells transfected with piGENE or FZD7-siRNA. HCT-116 cells were transiently transfected with piGENE or FZD7-siRNA. At 48 hours after transfection, 10 µg of cytosolic protein was prepared. Expression of β-catenin, Myc, Survivin and Jun were assessed. β-Actin served as a loading control.

Figure 4

Functional effects of FZD7-siRNA in HCT-116 and HT-29 cells. (A) Effect of FZD7-siRNA on cell viability. HCT-116 and HT-29 cells were transiently transfected with pcDNA3.1-U6 or pcDNA3.1-U6FZD7-siRNA. At 24 or 48 hours after transfection, HCT-116 and HT-29 cells were maintained in medium supplemented with G418. During 6 or 17 days after transfection, cell viability was tested by the MTS assay. (B) Effect of FZD7-siRNA on cell invasion. HCT-116 and HT-29 cells were transiently transfected with pcDNA3.1-U6 or pcDNA3.1-U6FZD7-siRNA. At 24 or 48 hours after transfection, an aliquot (10⁵ cells) of the prepared cell suspension was added into the upper chamber and cultured into the lower chamber filled with the culture medium containing fibronectin for 48 hours. Invasive cells were stained, and the average number of cells in five fields per membrane was counted in triplicate assay. (C) Effect of FZD7-siRNA on interferon response gene, OAS1. HCT-116 and Caco-2 cells were transiently transfected with piGENE or FZD7-siRNA. At 48 hours after transfection, total RNA were reverse-transcribed and amplified with primers specific for OAS1 as described in the Materials and Methods section. Poly(I:C) served as a positive control. OAS1 expression was normalized to GAPDH mRNA. Caco-2 served as a negative control.

Figure 5

Effect of FZD7-siRNA on apoptosis (A) and the mRNA expression level of FZD7 in primary CRC tissues (B). (A) Western blot analysis of apoptotic proteins in HCT-116 cells transfected with piGENE or FZD7-siRNA. HCT-116 cells were transiently transfected with piGENE or FZD7-siRNA. At 48 hours after transfection, 10 µg of cytosolic protein was prepared. Cleavage of caspase-3 and cytochrome c release were assessed. β-Actin served as a loading control. (B) The mRNA levels of FZD7 in primary CRC tissues and colon cancer cell lines were examined by real-time PCR. Data are presented as mean values and SD for three independent experiments compared with the level of GAPDH that is presented as 1.