Abstract 786: STAG2 acts as a transcriptional repressor and promotes invasion of muscle invasive bladder cancer cells

Abstract STAG2 (Stromal Antigen 2) functions in chromatid cohesion, DNA damage repair and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. We have previously found that in MIBC, loss of STAG2 protein expression is associated with better overall survival (n=169; p=0.049) and progression free survival (n=169; p=0.016). Based on these retrospective analyses, we hypothesized that STAG2 promotes an aggressive cell phenotype through its genomic interactions and transcriptomic regulation in MIBC. First, to study the effects of STAG2 on gene expression, we stably knocked down (KD) STAG2 in T24 MIBC cells using two short hairpin RNAs. Scrambled shRNA served as a control in all experiments. Second, using KD and control T24 cell lines, we performed RNA and chromatin-immunoprecipitation sequencing, then integrated these results utilizing the Cistrome analysis algorithm (Wang, S. et al., 2013) to determine how STAG2 regulates gene expression at its genomic binding sites. STAG2 KD led to the differential expression of 2158 genes, with 648 overlapping between the two shRNA cell lines. Through Cistrome analysis, we discovered that genes with increased expression after STAG2 KD were enriched for STAG2 binding sites (p=0.000503), yet genes with decreased expression did not show significant enrichment (p=0.873). This suggests that STAG2 functions as a transcriptional repressor, and subsequently we focused on genes with increased expression after STAG2 KD. We identified the significantly upregulated gene Reelin (RELN) (log2FC=2.89, 1.86; p<1*10-42, 1*10-17 for each shRNA, respectively), which has a well-established role in cell migration and invasion. To investigate this in vitro in MIBC cells, we performed time lapse microscopy and invasion assays to quantitatively determine cell movement over time. Compared to controls, T24 cells with STAG2 KD had reduced displacement (78 vs 114 µm, p<0.05), speed (0.30 vs 0.41 µm/min, p<0.05) and invasion (137 vs 190 cells/field, p<0.001) in vitro. Altogether, our results indicate that STAG2 functions as a transcriptional repressor and promotes movement and invasion of MIBC cells. This may explain why STAG2 protein loss leads to better outcomes for MIBC patients, and points to STAG2 protein expression as a potential prognostic biomarker. In our current work, we are utilizing a large drug screen of FDA-approved agents to identify vulnerabilities of tumor cells lacking STAG2. These studies will help to identify drugs that augment current standard of care and benefit the large proportion of patients with STAG2-mutant tumors. Citation Format: Sarah R. Athans, Nithya Krishnan, Swathi Ramakrishnan, Eduardo Cortes Gomez, Sofia Lage-Vickers, Monika Rak, Zara Kazmierczak, Aimee Stablewski, Kristopher Attwood, Jianmin Wang, Anna Woloszynska. STAG2 acts as a transcriptional repressor and promotes invasion of muscle invasive bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 786.