Endothelin-1 stimulated gene induction within colon cancer cells and fibroblasts and the effect of Endothelin receptor antagonism on key genes.

Samer-ul Haque1, Hazel Welch1, Michael Dashwood2, Xu Shiwen3, David Abraham3, Mo Heetun1, Noreen Farooqui1, Irving Taylor1, Marc Winslet1, Marilena Loizidou1. 1UCL, Dept. of Surgery, NW3 2QG, United Kingdom, 2UCL, Dept. of Clinical Biochemistry, NW3 2QG, United Kingdom, 3UCL, Centre for Rheumatology and Connective Tissue Diseases, NW3 2QG, United Kingdom.

Background: The vasoactive peptide ET-1 (Endothelin-1) contributes to colorectal tumourigenesis. At the cellular level, ET-1 promotes proliferation in cancer cells, via one of its two receptors (ETA). Additionally, ET-1 activates surrounding fibroblasts and may promote the creation of supporting tumour stroma.

Aim: To identify new genes that were up/down-regulated within cancer cells and fibroblasts post-ET-1 exposure and evaluated ETA & ETB receptor antagonism on gene expression.

Methods: Illumina micro-arrays determined differential gene expression post-ET-1 stimulation of 3 colorectal cancer cell lines and 4 human colon fibroblast strains isolated from areas adjacent to colorectal cancers. To confirm expression of genes of interest, we examined time point induction mRNA levels (RT-PCR; real-time RT-PCR). ETA (Zibotentan, BQ123) and ETB (BQ788) antagonistic effects were measured at the mRNA and protein levels (Immunoblotting). SiRNA was also used to confirm receptor involvement in regulation of these key genes.

Results: Four-hour ET-1 induction had a significant effect on gene up/down-regulation (p<0.01 + >1.5-fold) in all cancer cell lines (9 genes) and fibroblast strains (111 genes). We determined expression and effect of receptor antagonism of the following (table 1): (a) In cancer cells: (i) MT1X, maximum at 4hr, reversed by ETA antagonism; (ii) MMP7, late maximum induction (24hr) (undetectable by 4hr microarray), reversed by ETA antagonism; (iii)PPP2R5D, no significant up/down regulation, but levels were decreased by ETA antagonism. (b) In fibroblasts: (i) CTGF and (ii) ADM maximum at 2-4hr, both reversed by ETA & ETB antagonism; (iii) STC1, transient up-regulation (1hr) followed by down-regulation (4hrs in-line with 4hr microarray data), reversed by ETA & ETB antagonism.

ET-1 Regulated Genes

Table 1. ET-1 regulated genes and their associated roles in tumourgenesis.

Conclusions: ET-1 stimulates cancer cell and cancer-associated fibroblasts to produce signals that promote cancer growth and formation of tumour stroma. ETA and ETB receptor antagonists block a number of these signals. This study provides new evidence for the potential therapeutic use of Endothelin receptor antagonists as an adjuvant treatment for colorectal cancer.