Abstract 1406: FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer
Authors: Lu Yi-Tsung , Xu Tong , Hsieh Tien-Chan , Iqbal Maheen , Luo Zhifei , Farnham Peggy J. , Rhie Suhn K. , Goldkorn Amir 
Published: July 2021

Introduction: We previously reported that an aggressive subpopulation of highly tumorigenic, drug-resistant bladder cancer cells can arise from the bulk tumor cells without mutational events, and that this phenotypic plasticity is driven at least in part by epigenetic mechanisms. In the current work, we analyzed the chromatin accessibility of enhancers to identify transcription factors that contribute to this transition to a drug-resistant state.

Materials and methods: We used Hoechst nuclear staining and fluorescence activated cell sorting to isolate the Hoechst-low drug resistant side population (SP) cells from the less drug resistant Hoechst-high non-side population (NSP) cells in bladder cancer cell lines. We performed RNA sequencing and AcceSssIble assay to profile RNA expression, global DNA methylation and chromatin accessibility in SP and NSP cells. Putative enhancer regions were identified using H3K27ac ChIPseq data in bladder cancer cell lines, which are publicly available. We next performed transcription factor motif analysis on accessible enhancers near genes overexpressed in SP cells relative to NSP cells. The differentially expressed candidate target transcription factor by RNA-seq was validated using qRT-PCR. Functional validation was done using CRISPR knock out and drug resistance assays.

Results: A higher percentage of enhancers were differentially accessible between SP and NSP cells compared with the promoters. We identified 746 enhancers that are accessible only in SP cells and 465 enhancers only in NSP cells. Transcription factor motif analysis showed that FOX family motif was enriched in accessible enhancers near SP-overexpressed genes. Among FOX family transcription factors, FOXC1 was the only overexpressed transcription factor, and it was also the most significantly overexpressed gene in SP cells. FOXC1 was consistently overexpressed in bladder tumor tissues relative to normal bladder tissues in three independent studies in BaseSpace Correlation Engine. CRISPR knock-out of FOXC1 in multiple bladder cancer cell lines resulted in decreased cisplatin resistance.

Conclusion: We found that FOXC1 is overexpressed and its motif is enriched on the accessible enhancers when bladder cancer cells shift to a drug-resistant tumorigenic phenotype. Conversely, FOXC1 knock-out attenuates cisplatin resistance in bladder cancer cells. These observations suggest that differential expression and enhancer binding of FOXC1 promotes the previously observed, mutation-independent shift towards cisplatin resistance in bladder cancer.

Citation Format: Yi-Tsung Lu, Tong Xu, Tien-Chan Hsieh, Maheen Iqbal, Zhifei Luo, Peggy J. Farnham, Suhn K. Rhie, Amir Goldkorn. FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1406.