Down-regulation of frizzled-7 expression decreases survival, invasion and metastatic capabilities of colon cancer cells

Abstract

Background: The canonical Wnt signalling pathway is activated in most sporadic colorectal cancers (CRCs). We previously reported that FZD7 functions as a receptor for the canonical Wnt signalling pathway in colon cancer cells.

Methods and results: In this study, we examined the function of FZD7 in survival, invasion and metastatic capabilities of colon cancer cells. FZD7_siRNA transfection decreased cell viability of HT-29 and HCT-116 colon cancer cells. Expression of c-Jun, phosphorylation of JNK and c-Jun, and activation of RhoA were suppressed after FZD7_siRNA transfection into HCT-116 cells. In vitro invasion activity and Wnt target gene expression were also reduced in HCT-116 cells transfected with FZD7_siRNA. Liver metastasis of stable FZD7_siRNA HCT-116 cell transfectants in scid mice was decreased to 40-50% compared to controls. The mRNA levels of FZD7 in 135 primary CRC tissues were examined by real-time PCR. FZD7 mRNA levels were significantly higher in stage II, III or IV tumours than in non-tumour tissues (P<0.005), and overall survival was shorter in those patients with higher FZD7 expression (P<0.001).

Conclusion: These data suggest that FZD7 may be involved in enhancement of survival, invasion and metastatic capabilities of colon cancer cells through non-canonical Wnt signalling pathways as well as the canonical pathway.

Figure 1

Preparation and selection of FZD7_siRNA. (A) Real-time PCR analysis of FZD7 mRNA expression in HCT-116 cells transfected with shRNA expression vectors harbouring siRNA against FZD7. Thirteen siRNAs were designed based on the nucleotide sequence of FZD7 gene. HCT-116 cells were transiently transfected with shRNA expression vectors. At 48 h after transfection, total RNAs were reverse-transcribed and the levels of mRNA expression of FZD7 were measured by real-time PCR. (B) FZD7_siRNA8 specifically inhibited the expression of FZD7. 293T cells were transiently transfected with various combinations of pEF4, pEF4-FZD7-V5, pEF4-FZD1-V5, scramble RNA and FZD7_siRNA8. Cell lysates (10 μg) were analysed by western blotting using anti-V5 or anti-β-actin antibodies.

Figure 2

FZD7_siRNA suppressed cell viability and invasion. (A) Effect of FZD7_siRNA on cell viability. HCT-116 and HT-29 cells were transiently transfected with FZD7_siRNA or scramble siRNA control. At 6 days after transfection, HCT-116 and HT-29 cells were stained with crystal violet. Cell viability was determined by absorbance at 595 nm. (B) Effect of FZD7_siRNA on cell cycle. HCT-116 cells were transfected with FZD7_siRNA or scramble siRNA. Cells were harvested 48 h after transfection and cell-cycle analysis was performed using a Cytomics FC500 cell sorter. (C) Effect of FZD7_siRNA on MAP kinase protein expression. HCT-116 cells were transiently transfected with FZD7_siRNA or scramble siRNA. At 48 h after transfection, cytosolic protein (10 μg) was analysed. Expression of p38, phospho-p38, ERK, phosho-ERK, JNK, phospho-JNK, c-Jun or phospho-c-Jun was assessed by western blot analysis. β-Actin was used as a loading control. Ratio of band intensity indicates the ratio of band intensity of tested protein to that of β-actin. Band intensity was measured by using ImageJ software. (D) Effect of FZD7_siRNA on RhoA activation. HCT-116 cells were transfected with FZD7_siRNA or scramble siRNA. At 48 h after transfection, GST-RhoA expression was assessed. Total RhoA was used as a loading control. Ratio of band intensity indicates the ratio of band intensity of tested protein to that of β-actin. (E) Effect of FZD7_siRNA on cell invasion. HCT-116 cells were transiently transfected with FZD7_siRNA or scramble siRNA. At 24 h after transfection, an aliquot (10⁵ cells) of the prepared cell suspension was added into the upper chamber, the lower chamber was filled with culture media containing fibronectin and cultured for 48 h. Invasive cells were stained and the average number of cells in five fields per membrane was counted in triplicate. (F) Effect of FZD7_siRNA on Wnt target gene expression. HCT-116 cells were transiently transfected with FZD7_siRNA or EGFP_siRNA. At 48 h after transfection, total RNAs were reverse-transcribed and the level of mRNA expression of Wnt target genes was measured by real-time PCR.

Figure 3

FZD7_siRNA inhibits in vivo metastasis. (A) The level of mRNA expression of FZD7, MT1-MMP and Jun in stable transfectants expressing FZD7_siRNA. HCT-116 cells were transfected with shRNA-expressing vectors and stable transfectants were selected by neomycin resistance. The expression levels of FZD7, MT1-MMP and Jun mRNAs in transfectants were assayed by real-time PCR. (B) Tcf-reporter transcriptional activity in FZD7_siRNA transfectants. Stable transfectants were transiently co-transfected with TOPflash reporter plasmid and pRL-TK plasmid encoding Renilla luciferase as an internal control for transfection efficiency. At 48 h after transfection, cell lysates were measured for relative luciferase activities. Data are presented as mean values ±s.d. for three independent experiments and compared with the level of luciferase activity obtained in the presence of stable transfectants expressing siRNA against EGFP that is represented as 1. (C) Cell viability of FZD7_siRNA transfectants. Stable transfectants were seeded at 10⁴ cells per well in 2 ml medium in a six-well tissue culture plate. At 8 days after seeding, cells were stained with crystal violet. Cell viability was determined by absorbance measurements at 595 nm using 2030 ARVO × 4 spectrophotometer. (D) Invasive ability of FZD7_siRNA transfectants. Aliquots (10⁵ cells) of the prepared cell suspension were added into the upper chamber and cultured into the lower chamber filled with culture media containing fibronectin for 48 h. Invasive cells were stained and the average number of cells in five fields per membrane was counted in triplicate assay. (E) In vivo metastatic ability of FZD7_siRNA transfectants. Stable transfectants expressing FZD7_siRNA or EGFP_siRNA were injected into the spleen of scid mice. Three weeks after transplantation mice were killed to count the number of liver metastasis.

Figure 4

The expression level of FZD7 mRNA in primary colorectal tumour and non-tumour tissues. (A) Comparison of tumour with non-tumour tissues. The mRNA levels of FZD7 in primary colorectal tumour and non-tumour tissues were examined by real-time PCR. Tumours were divided into four groups according to the pathological stage (see Table 1). (B) Effect of clinical course after surgery on FZD7 expression level. Patients were divided into disease free after surgery and recurrence or death after surgery groups according to the follow-up information after surgery. Disease free indicated a patient group with no recurrence after surgery. Recurrence+Death indicated a patient group with recurrence or death after surgery. (C) Kaplan–Meier analysis for overall survival of patients. High or low indicates the patients with the FZD7 mRNA levels ⩾ or < the mean value (11.1) of all tumours tested.