Abstract 2916: TAF15 as an important mediator of RNA polymerase II-dependent drug tolerance
Authors: Kume Kohei , Ito Kohei , Iwaya Takeshi , Nishizuka Satoshi S. 
Published: July 2016

Background: Cancer relapse after chemotherapy is thought to originate from drug-tolerant cancer cell subpopulations. We studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Previous DTC formation-based compound screening identified an RNA polymerase (RNAP) II inhibitor, α-amanitin (α-AMA), suggesting the existence of an RNAPII-dependent drug tolerance in cancer cells. However, the mechanism underlying this regulation is unclear.

Materials and Methods: DTCs derived from the gastric cancer cell line MKN45 were used as an in vitro model for gastrointestinal tumor recurrence. For the generation of the DTCs and those of untreated colonies (i.e., control colonies), cells were sparsely disseminated (100 cells/cm2) into the presence or absence of cisplatin at 50% colony inhibitory concentration. To identify the DTC formation responsible genes, a DNA microarray analysis was performed followed by gene ontology analysis and quantitative RT-PCR (qRT-PCR) validation. For gene knockdown experiments with those that have been identified as DTC-formation responsible genes, cells were treated with small interference RNAs (siRNAs) for 48 hrs and then sparsely disseminated into the microplates in the presence of cisplatin for the DTC formation.

Results: In terms of the RNAPII-dependence of the DTC formation, we focused on RNAPII-binding protein-coding genes in the list of the top 2.5% of specifically induced genes in DTCs. The transcriptional screening identified only one gene, TATA-binding protein-associated factor 15 (TAF15). The level of TAF15 mRNA was 2.1-fold higher in DTCs than in the control colonies. A protein family consisting of fusion in sarcoma (FUS), Ewing's sarcoma (EWS), and TAF15 is called the FET (FUS/EWS/TAF15) family, and they play various roles in transcription as well as RNA processing. Among the FET family proteins, the TAF15 protein level was specifically increased in DTCs, suggesting that TAF15-specific function may contribute to DTC formation. Next, we examined whether TAF15 protein depletion by siRNA affects DTC formation. The TAF15 depletion preferentially inhibited DTC formation despite the fact that the degree of inhibition to the control colony formation was limited. With an anti-TAF15 siRNA treatment, the original small, round-shaped MKN45 cells exhibited spindle-like morphological changes within 48 hrs. Interestingly, α-AMA treatment also induced similar morphological changes, suggesting that RNAPII inhibition caused TAF15 depletion. Consistent with the morphological changes, both TAF15 mRNA and protein levels decreased in response to α-AMA treatment.

Conclusion: These results suggest that TAF15 is an important mediator of RNAPII-dependent drug tolerance and one of the potential molecular therapeutic targets of α-AMA in the context of cancer relapse after chemotherapy.

Citation Format: Kohei Kume, Kohei Ito, Takeshi Iwaya, Satoshi S. Nishizuka. TAF15 as an important mediator of RNA polymerase II-dependent drug tolerance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2916.