Mutations In TERT Promoter Have A Prominent Role In Cancer Etiology

Introduction

Somatic aberrations in the genome play a vital role in cancer development, protein-coding sequence accounts for less than 2% in the total genome content. If the cancer genome needs to be well understood, investigations aiming at dissecting the role of non-coding mutation are indispensable. Reports have shown that mutation in the promoter region could either create new banding sites or disrupt transcription factor binding sites (TFBS) for transcription.

Material and method

A Transcription library was constructed through cell-free expression platform in our lab. The library was used to screen with WT/MUT TERT promoter oligos. In different cell lines, the binding events were validated by promoter activity assay and ChIP-seq. Candidate TFs were knocked down by means of siRNA, total RNA was collected and send for mRNA profiling. Profiling data was validated by RT-PCR and western blot. By the means of CRISPR, the candidate TF was knocking out in MCC26 cell lines (mutation in TERT promoter). Cell cycle analysis, proliferation assay were used to validate the related pathway.

Results and discussion

In this project, we found two promising ETS family transcription factors (TF1 and TE3) preferentially binding to mutant TERT promoter. We found the cell cycle regulation to be affected by downregulation of TERT and the selected transcription factor from siRNA-induced silencing, cell cycle analysis(FACS) and cell proliferation assay were performed for validation.

Conclusion

Based on the hypothesis the mutations in TERT promoter core region could generate a novo ETS transcription factor binding site, we screen the promoter oligos with self-made cell-free expression transcription factors library, we get two TFs candidates, especially from ETS family members, could bind to the mutation type of TERT promoter, providing a plausible handle for studying the mechanism of TERT reactivation, which if targeted could have immense therapeutic implications.