UC San Diego
Defining the Cellular Consequences of Loss of the Cohesin Complex Subunit STAG2 in Cancer Cells
- Author(s): Richardson, Amelia Katherine
- Advisor(s): Desai, Arshad
- et al.
Inactivating mutations in the cohesin subunit STAG2 are frequent in many cancers, but the consequences of these mutations have not been clearly defined. STAG2 loss may impair sister chromatid cohesion, but it is not clear if it also increases aneuploidy as initially proposed. Alternatively, STAG2 loss may contribute to cancer through defective DNA repair and genomic instability or through aberrant transcription, which could dysregulate cancer-regulating genes. The primary goal of this project is to thoroughly define the effects of STAG2 loss on cohesin’s cellular functions to better understand why STAG2 is frequently lost in cancers.
To address this goal, I developed a quantitative assay to measure cohesion strength in live cells. Cohesion fatigue timing indicated cohesion strength after STAG1 or STAG2 perturbation. I also measured the effect of STAG1 and STAG2 perturbation on inter-kinetochore stretch and anaphase defects, both indicators of cohesion strength. Surprisingly, collective results suggested that STAG1 and STAG2 are functionally redundant for cohesion strength in immortalized hTERT RPE-1 cells, and that STAG2 loss does not impair cohesion in glioblastoma H4 or colorectal cancer HCT116 cells as previously reported.
I then quantified the effect of STAG2 depletion on DNA repair of localized DSBs using a reporter assay for HR and NHEJ and found that STAG2 depletion does not alter HR or NHEJ frequency in U-2 OS cells. Loss of STAG2 also does not severely sensitize H4 (or hTERT RPE-1 and HCT116) cells to PARP inhibition as previously reported.
Finally, I assessed the effect of STAG1 and STAG2 perturbation on gene expression by RNA-seq in hTERT RPE-1 cells and Ewing sarcoma A673 cells. There, I found the exiting result that STAG1 depletion and STAG2 depletion have differential effects on gene expression in Ewing sarcoma cells, but not in other tested cell lines. These preliminary results suggest a unique potential role for STAG2 loss in the aberrant gene expression that drives Ewing sarcoma.
A secondary, collaborative goal of this project was to clarify the immediate cellular consequences of aneuploidy. I confirmed, through live imaging of mis-segregating chromatin, that a mitotic timer, and not mis-segregation, is responsible for subsequent cell-cycle arrest.