Abstract 4019: Regulatory role of Gli motifs in thymidylate synthase expression .
Authors: Pochi Subbarayan R. , Doshi Nikesh , Han Xiaoqing , Wang Zhiqiang , Capobianco Anthony , Robbins David , Ardalan Bach 
Published: August 2014

Fluoropyrimidine analogue 5-Flurorouracil (5-FU) is the mainstay of CRC treatment. It was designed as a competitive inhibitor of the enzyme thymidylate synthase [TS; TYMS]. All the new agents (Irinotecan, CPT-11, Avastin etc) are effective only in combination with 5-FU. The enzyme thymidylate synthase is pivotal for DNA replication. It controls cell survival and proliferation. Increases in TS level that follows 5-FU administration renders the treatment ineffective. Considerable efforts have been made to reverse 5-FU resistance by down-regulating TS expression. In this context, we demonstrated that arsenic trioxide (ATO) sensitized 5-FU resistant colorectal cancer cells by inhibiting TS mRNA expression both in vitro and in clinic. However, the precise mechanism of TS reduction by arsenic trioxide is not known.

Many investigators, including ourselves, have shown a direct correlation between Glioma associated oncogene homolog (Gli) and TS expression in cultured cells as well in patient samples, who received arsenic trioxide and 5-FU combination therapy. However, the link between ATO, Gli and TS is not known.

Gli is an important molecule in hedgehog (hh) signaling. It plays a vital role in development and cancer. As a transcription factor, Gli binds the sequence 5’ GACCACCCA 3’ a conserved 9 bp DNA motif. We hypothesized that TS promoter may harbor one or more of the 9 bp Gli motifs.

We queried for Gli motifs in the TS gene regulatory region. Position specific base frequencies for the TF consensus sequence for Gli1-3, Ci, cECF, TGM were prepared using the data from Hallikas et al. (2006). We used FIMO (Find Individual Motif Occurrences) tool in the Meme Suite (http://meme.nbcr.net) to search for Gli motifs in the query sequence. Anti-Gli antibodies and non-specific IgG were used to precipitate chromatin immuno complex. The immuno precipitates were probed with TS specific primer.

This search yielded 35 possible Gli motifs in TS promoter. From among the 35 motifs with significant homology (p≤0.001), only top eight matches had passed FDR threshold (q≤0.05). However, these eight matches represented three distinct binding sites on TS promoter. Thus we computationally determined that TS promoter has three unique Gli motifs viz BS1, BS2 and BS3. BS-2 in TS promoter is identical to BS-3 on the Jun and BS1 is identical to the BS 1 of Fgf15 which had been experimentally confirmed to be regulated by Gli1. The binding site was subsequently confirmed by ChIP assays.

It appears that the inhibition of Gli1 by ATO down regulates TS expression. This cascade of reactions initiated by ATO reverts refractory tumors, once again sensitive to 5-FU. Taken together the results strongly indicate a direct role of Gli in TS transcription.

Citation Format: Subbarayan R. Pochi, Nikesh Doshi, Xiaoqing Han, Zhiqiang Wang, Anthony Capobianco, David Robbins, Bach Ardalan. Regulatory role of Gli motifs in thymidylate synthase expression . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4019. doi:10.1158/1538-7445.AM2013-4019