HOXB13 is downregulated in colorectal cancer to confer TCF4-mediated transactivation

Abstract

Mutations in the Wnt signalling cascade are believed to cause aberrant proliferation of colorectal cells through T-cell factor-4 (TCF4) and its downstream growth-modulating factors. HOXB13 is exclusively expressed in prostate and colorectum. In prostate cancers, HOXB13 negatively regulates beta-catenin/TCF4-mediated transactivation and subsequently inhibits cell growth. To study the role of HOXB13 in colorectal tumorigenesis, we evaluated the expression of HOXB13 in 53 colorectal tumours originated from the distal left colon to rectum with their matching normal tissues using quantitative RT-PCR analysis. Expression of HOXB13 is either lost or diminished in 26 out of 42 valid tumours (62%), while expression of TCF4 RNA is not correlated with HOXB13 expression. TCF4 promoter analysis showed that HOXB13 does not regulate TCF4 at the transcriptional level. However, HOXB13 downregulated the expression of TCF4 and its target gene, c-myc, at the protein level and consequently inhibited beta-catenin/TCF-mediated signalling. Functionally, forced expression of HOXB13 drove colorectal cancer (CRC) cells into growth suppression. This is the first description of the downregulation of HOXB13 in CRC and its mechanism of action is mediated through the regulation of TCF4 protein stability. Our results suggest that loss of HOXB13 may be an important event for colorectal cell transformation, considering that over 90% of colorectal tumours retain mutations in the APC/beta-catenin pathway.

Figure 1

Expression pattern of HOXB13 mRNA in colorectal tumours. Real-time RT–PCR was performed with RNA extracted from colorectal tumours and matching normal tissues. Levels of HOXB13 in both normal and tumour tissues were normalised by β-actin. Relative expression of HOXB13 in normal tissues were averaged (black bar), which was used for comparative HOXB13 expression in each tumours. Dotted line represents 95% confidence interval for the range of HOXB13 expression in normal colorectum. ^*HOXB13 not detectable in tumours. Note X-axis is a log scale.

Figure 2

HOXB13 disrupts β-catenin/TCF-mediated signals. (A) Real-time RT–PCR was performed to show baseline levels of HOXB13 in established colorectal tumour cell lines. Normal rectum and LNCaP prostate cancer cells were used as references. (B) Cells were transiently transfected with 100 ng of pIRES-TCF4RE-Luc, 20 ng of pCMV-β-gal, and 100 ng of pFLAG-HOXB13 with or without 10 nM R1881. pFLAG-CMV was used as a counterpart of pFLAG-HOXB13. ^*P<0.05. (C) CV-1 cells were transfected with either pGL-TATA-luc or pBV-myc-luc and pIRES-HOXB13. ^*P=0.02.

Figure 3

HOXB13 suppresses expression of TCF4 at the post-transcriptional level. (A) Cells were infected with either Ad-GFP or Ad-GFP-HOXB13 virus (100 MOI) for 48 h. Nuclear proteins were separated by gel electrophoresis followed by Western blot analysis. (B) Potential promoter region of TCF4 was cloned into pGL-luc system (pGL-TCFp-luc) followed by transient transfection assay. (C) Cells were transfected with pGL-TCF4p-luc and either pFLAG or pFLAG-HOXB13. ^*P>0.2; ^**P<0.05. (D) HCT116 cells were infected with Ad-GFP or Ad-GFP-HOXB13 (100 MOI) followed by extraction of total RNA. Real-time RT–PCR was performed for TCF4 expression and evaluated by standard curve methods. RNA from normal rectum was used to draw the standard curve for TCF4 and β-actin. ^*P=0.32.

Figure 4

HOXB13 inhibits the proliferation of HCT116 colon cancer cells. (A) Ad-HCT116 cells were infected with either Ad-GFP-HOXB13 or Ad-GFP viruses (10 MOI). GFP-expressing cells were monitored under the fluorescence microscope. (B) The same study was performed as in (A). At indicated days after virus infection (1 MOI), HCT116 cells were stained with MTT reagent and optical density at 570 nm was measured.