PhD Defense | Auchi Inalegwu | Comprehensive transcriptome mining for biomarkers of radiation sensitivity in breast cancer cells: specific focus on circRNA regulated networks
Comprehensive transcriptome mining for biomarkers of radiation sensitivity in breast cancer cells: specific focus on circRNA regulated networks
Radiotherapy is the standard of care for breast cancer. However, surviving radioresistant cells can repopulate following treatment and provoke relapse. Better understanding of the molecular mechanisms of radiation resistance may help to improve treatment of radioresistant tumours.
To emulate radiotherapy at the cellular level, human MCF7 breast cancer cells were exposed to daily radiation doses of 2 Gy up to an accumulated dose of 20 Gy. The fractionally irradiated cells (FIR20) were then compared with the naïve parental (PAR) and age-matched controls (AMC) using phenotypic assays followed by transcriptomic profiling of mRNA and circular RNA (circRNA) by RNA-sequencing.
The cell lines were characterized at baseline (chapter 4) and following subsequent single dose irradiation (chapter 5). Cell proliferation, radiosensitivity, tamoxifen cytotoxicity, cell cycle analysis, and DNA damage response were evaluated using live cell imaging, clonogenic survival assay and immunocytochemistry. RNA-seq was used for whole transcriptome analysis and the results were validated by real-time quantitative polymerase chain reaction, immunocytochemistry, and/or western blot. Dysregulated pathways were identified by gene set enrichment analysis and a competitive endogenous RNA (ceRNA) network of the circRNA-miRNA-mRNA interactions associated with radioresistance was constructed by integrating the RNA-seq results with in silico target prediction analysis.
FIR20 cells displayed increased radioresistance and population doubling time, reduced accumulation of DNA damage response proteins following irradiation, and a core radioadaptive signature of significantly differentially expressed mRNAs (DEMs) and circRNAs (DECs). Furthermore, FIR20 cell transcriptome overlapped significantly with canonical radiation response signatures and basal-like breast cancer subtype and exhibited remarkable commonality with endocrine therapy resistance gene signatures. Cross resistance of FIR20 cells to tamoxifen was confirmed in vitro. Predictive and functional enrichment analysis revealed a circRNA-governed, gene-regulatory network that promotes stemness and inflammatory signaling in FIR20 cells. Moreover, high expression of many core radioadaptive signature genes were associated with poorer breast cancer survival outcome and tumor stage in patients who received radiotherapy, endocrine therapies, and chemotherapy, and additionally, exhibited a stable regulatory pattern after acute irradiation in vitro.
In conclusion we established for the first time a comprehensive, high confidence ceRNA network that governs the radioadaptive response of radioresistant MCF7 breast cancer cells. We identified several potential gene and circRNA biomarkers of radioresistance which also likely mediate resistance to tamoxifen and other systemic therapies. We propose that the transcriptional signatures identified in this study are potential promising candidates for further functional analysis that could also serve as therapeutic biomarkers and targets for more efficacious combination therapies.
Winnok De Vos (UANTWERPEN)
Kris Laukens (UANTWERPEN)
SCK CEN mentors:
Roel Quintens (SCK CEN)
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