STAG2 (Stromal Antigen 2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (n=119 bladder cancer clinical samples), we found several STAG2 mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (n=346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for MIBC patients. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth, whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy number variations, or cell cycle distribution. However, unbiased RNA sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion in vitro. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a non-canonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in STAG2-mutant MIBC.

<p>Supplementary methods, Supplementary Figures S1-S3, and Supplementary tables</p>

<div><p>Stromal antigen 2 (STAG2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle-invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (<i>n</i> = 119 bladder cancer clinical samples), we found several <i>STAG2</i> mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (<i>n</i> = 346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for patients with MIBC. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth<i>,</i> whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy-number variations, or cell-cycle distribution. However, unbiased RNA-sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion <i>in vitro</i>. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a noncanonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in <i>STAG2</i>-mutant MIBC.</p>Significance:<p>The cohesin component STAG2 regulates cell motility and invasion. STAG2 expression is associated with decreased MIBC survival and may be a useful biomarker to guide bladder cancer treatment.</p></div>

<div><p>Stromal antigen 2 (STAG2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle-invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (<i>n</i> = 119 bladder cancer clinical samples), we found several <i>STAG2</i> mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (<i>n</i> = 346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for patients with MIBC. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth<i>,</i> whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy-number variations, or cell-cycle distribution. However, unbiased RNA-sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion <i>in vitro</i>. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a noncanonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in <i>STAG2</i>-mutant MIBC.</p>Significance:<p>The cohesin component STAG2 regulates cell motility and invasion. STAG2 expression is associated with decreased MIBC survival and may be a useful biomarker to guide bladder cancer treatment.</p></div>

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&lt;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&lt;0.05), speed (0.30 vs 0.41 µm/min, p&lt;0.05) and invasion (137 vs 190 cells/field, p&lt;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.