The effect of distinct BRCA1 splice forms on drug sensitivity in breast- and ovarian cancer cell lines
Germline mutations in BRCA-genes cause higher risk of cancer development, particularly in breast and ovaries. PARP inhibitors are a promising treatment option for carriers of BRCA1/2 mutations diagnosed with these tumor types. Pathogenic spliceogenic variants have been found in BRCA1 that change the expression of BRCA1 transcripts, in which the expression of shorter BRCA1 transcripts (BRCA1 ∆11 and ∆11q) increases, but expression of full-length BRCA1 transcript (BRCA1-FL) decreases. However, additional work is needed to determine how different BRCA1 transcripts can affect the drug sensitivity in breast- and ovarian cancer. The aim of this study was to knock out BRCA1 expression in the T47D breast cancer cell line and the CAOV3 ovarian cancer cell line and overexpress longer and shorter transcripts of BRCA1 in the cells. Subsequently, the aim was to assess their sensitivity to PARP inhibition and thereafter, study how each BRCA1 splice form affects the drug sensitivity of cancer cells. Finally, the aim was to use bioinformatic analysis with RNA sequencing and proteomics data to identify phenotypical changes in T47D cell lines with different expression of BRCA1 variants. CRISPR/Cas9 technique was used to knock out (KO) BRCA1 expression in T47D and CAOV3. The knockout of the gene was confirmed with RT-qPCR and western blot (WB) in T47D cells, but the BRCA1 knockout in CAOV3 was unsuccessful. Therefore, only T47D cell line was used in the project to further explore the drug sensitivity to the PARP inhibitor olaparib. Drug sensitivity was studied by clonogenic assay, and treatment with olaparib demonstrated that cells with loss of BRCA1 expression are more sensitive to PARP inhibitors than cells expressing wildtype (WT) BRCA1. The clonogenic assay also revealed that cells with BRCA1 loss grew slower than control cells. Subsequently, the BRCA1-FL transcript and the shorter transcript BRCA1∆11q were overexpressed in T47D BRCA1-KO cells using lentiviruses. RT-qPCR and WB results showed 3- and 7-fold expression of the BRCA1 splice forms in BRCA1-FL and BRCA1∆11q transduced cells, respectively, compared to control cells. To examine how different BRCA1 splice forms affect the drug sensitivity of breast cancer cells, the cells were treated with olaparib. Results revealed that drug sensitivity was partially reserved when both FL and ∆11q splice forms were overexpressed in T47D cells. RNA sequencing and proteomics data on cells with different splicing in BRCA1 exon 11 showed changed RNA and protein expression with decreased BRCA1-FL expression and increased BRCA1∆11/∆11q transcripts. Moreover, changes in gene regulatory pathways such as DNA repair were also observed. This study demonstrated that loss of BRCA1 expression sensitizes cells to PARP inhibition and that drug sensitivity is partially reserved when different splice variants of BRCA1 are overexpressed in T47D cells. Also, that changed expression of BRCA1 splice variants affects the phenotype of breast cancer cells. Future studies will focus on using the T47D cell model to study further the role of BRCA1 splice forms in breast cancer cells and explore how BRCA1 splice forms affect the phenotype and drug sensitivity of ovarian cancer by using similar methods.
Lífeindafræði, Stökkbreytingar, Krabbamein
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