Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy

Abstract

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.

Figure 1

Distribution of single nucleotide variants identified in the discovery screen through exome sequencing: total (a), according to type of nucleotide substitution (b), and according to the predicted effect (c).

Figure 2

Distribution of mutations in genes recurrently mutated in UBC that are expressed in >30% of tumors; joint analysis of the discovery and prevalence screens. In 22/77 tumors (28.6%), none of the genes listed in this Figure was found to be mutated.

Figure 3

Immunohistochemical analysis of STAG2 expression in bladder tumors of different stage and grade: 2 of the tumors show lack of STAG2 expression (a, c) and one tumor shows strong expression (b). Of note the strong STAG2 protein expression in normal urothelium of a patient with a STAG2-negative tumor (a) and in the stroma of all tumor samples. (T, tumor; S, stroma; NU, normal urothelium). Scale bar: 200 µm.

Figure 4

STAG2 loss is not associated with aneuploidy in primary tumors and in 639V bladder cancer cells. Effects of STAG2 reconstitution on cell growth. (a) SNP array genomic plots show lack of chromosomal changes (aneuploidy) in a tumor lacking STAG2 expression (note strong STAG2 labeling of normal stroma). Scale bar: 200 µm. (b) Western blotting analysis of STAG2 in UBC lines shows undetectable expression in 4 of 11 lines used for functional studies. (c) STAG2 overexpression in UM-UC-6 cells leads to reduced colony formation efficiency. (d) Efficient STAG2 knockdown in 639V cells demonstrated by western blotting does not lead to consistent changes in chromosome number (quantification shown in Supplementary Table 12).

Figure 5

Kaplan-Meier plots of the association of STAG2 expression with outcome in patients with UBC. (a) Recurrence in patients with STAG2-high (n=309) vs. STAG2-low (n=171) NMIBC. (b) Progression in patients with STAG2-high (n=309) vs. STAG2-low (n=171) NMIBC. (c) Progression in patients with STAG2-high (n=158) vs. STAG2-low (n=24) MIBC. (d) Cancer-specific survival of patients with STAG2-high (n=158) vs. STAG2-low (n=24) MIBC. P-values correspond to the results of the multivariable analysis. Details on results and variables used for adjustment are shown in Supplementary Tables 17–20.