Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcaemic type

Abstract

Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a lethal and sometimes familial ovarian tumour of young women and children. We and others recently discovered that over 90% of SCCOHTs harbour inactivating mutations in the chromatin remodelling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually exclusive ATPases of the SWI/SNF chromatin remodelling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry in more than 3000 primary gynaecological tumours. Among ovarian tumours, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high-grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumour cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4-negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumours, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase inhibitor trichostatin A. Re-expression of SMARCA4 or SMARCA2 inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2, is highly sensitive and specific for SCCOHT and that restoration of either SWI/SNF ATPase can inhibit the growth of SCCOHT cell lines.

Keywords: HDAC inhibitor; SMARCA2/BRM; SMARCA4/BRG1; SMARCB1/INI1; SWI/SNF; epigenetic silencing; hypercalcaemic type; rhabdoid tumour; small cell carcinoma; trichostatin A.

Figure 1

Immunohistochemical analysis of core SWI/SNF proteins in SCCOHT. (A) Dual loss of SMARCA4/BRG1 and SMARCA2/BRM in SCCOHT. Endothelium and lymphocytes are internal positive controls for both proteins. Arrows denote rare tumour cells expressing SMARCA2. SMARCB1/INI1 protein expression serves as a positive control for tumour cell immunoreactivity. (B) SMARCA2/BRM expression and subcellular localization in regions of small cell versus large cell morphology. When present, SMARCA2 only showed nuclear localization in the small cells (arrows, A). In contrast, scattered tumour cells with large cell morphology also expressed SMARCA2 in the cytoplasm (arrowheads, B). (C) SMARCA4/BRG1 and SMARCA2/BRM expression in normal ovary from premenopausal women. SMARCA4 was expressed strongly in oocytes and granulosa cells but was either weak or absent in stromal cells. In contrast, SMARCA2 showed diffuse and strong expression in oocytes, granulosa cells, and stromal cells. In addition, both proteins showed uniform expression in ovarian surface epithelium (data not shown). H&E and SMARCA4 immunohistochemistry images are reproduced from our previous publication, Ramos et al 2 (Supplementary Figure 4, panels A–D).

Figure 2

Immunohistochemical analysis of SMARCA4/BRG1 and SMARCA2/BRM in ovarian clear cell carcinoma. Tumours were negative for either SMARCA4 (middle panels) or SMARCA2 (bottom panels) or showed intact expression of both proteins (upper panels). No dual deficient tumours were identified. Similar to SCCOHT, rare scattered SMARCA2‐positive cells (arrows) were identified in some SMARCA2‐negative tumours.

Figure 3

Immunohistochemical analysis of SMARCA4/BRG1 and SMARCA2/BRM in endometrial stromal sarcoma (ESS) and dedifferentiated carcinoma (DDC) of the uterus. Three uterine tumour types showed SMARCA4/BRG1 deficiency: low‐grade ESS (LGESS, n = 2, upper panels), high‐grade ESS (HGESS, n = 2, middle panels), and DDC (n = 1, lower panels). Both cases of SMARCA4‐negative LGESS maintained expression of SMARCA2. In contrast, both HGESS and the DDC that lacked SMARCA4 were also deficient in SMARCA2.

Figure 4

SMARCA2 silencing in SCCOHT tumours and cell lines and reactivation by the histone deacetylase inhibitor trichostatin A. (A) Heat map depicting the expression of genes encoding SWI/SNF family members in SCCOHT tumours (SCCO‐002, ‐012, ‐014, and ‐015), patient‐derived mouse xenografts (PDX‐040 and −065), and SCCOHT cell lines (BIN67 and SCCOHT1). SMARCA2 mRNA is strongly down‐regulated in contrast to almost all other SWI/SNF genes. Premenopausal ovaries were used as a reference. (B) Real‐time PCR of steady‐state SMARCA4 and SMARCA2 mRNA levels in SCCOHT cell lines (BIN67 and SCCOHT1) compared with other ovarian cell lines. SVOG3e: immortalized granulosa cells; KGN: adult granulosa cell line; NOY1: yolk sac tumour cell line. Levels of mRNA were normalized to GAPDH. (C) Western blotting for SMARCA4 and SMARCA2 in SCCOHT cell lines. Vinculin served as a loading control. Both SCCOHT cell lines expressed SMARCB1/INI1 (data not shown), similar to primary tumours (Figure 1A). (D) Specific up‐regulation of SMARCA2/BRM protein by the histone deacetylase inhibitor trichostatin A (TSA). BIN67 cells were treated for 72 h with TSA, GSK343, trametinib or 5‐azacytidine, and SMARCA2 expression was analysed by western blotting. Ctrl denotes vehicle treatment. SMARCA2 (lane 2) denotes cells transduced with lentivirus expressing SMARCA2. KGN: adult granulosa cell tumour cells. Ponceau S staining served as a loading control. (E) Real‐time PCR of SMARCA4 and SMARCA2 mRNA levels in BIN67 cells after 72 h of treatment with 0.6 µm TSA. Although absolute baseline SMARCA2 mRNA levels are significantly lower than SMARCA4 (Figure 4B), both are arbitrarily set to 1 in this experiment. SMARCA2 mRNA levels are strongly up‐regulated by TSA compared with SMARCA4. (F) SMARCA2 protein up‐regulation is TSA‐dose‐dependent in SCCOHT cell lines. BIN67 and SCCOHT1 cells were treated with increasing doses of TSA for 72 h, and SMARCA2 expression was analysed by western blotting. Ctrl denotes vehicle treatment. SMARCA2 (lane 2 for each cell line) denotes cells transduced with lentivirus expressing SMARCA2. ES‐2: ovarian endometrioid adenocarcinoma cells. Ponceau S staining served as a loading control.

Figure 5

Re‐expression of SMARCA4 or SMARCA2 suppresses SCCOHT cell growth. (A) BIN67 cells were transduced with lentiviruses expressing GFP, SMARCA4 or SMARCA2 and seeded at a density of 4000 cells per well in 96‐well plates for monitoring cell growth using an IncuCyte ZOOM® live cell monitor. The percentage of confluence in each well was calculated and plotted to determine the effect of SMARCA4 or SMARCA2 re‐expression on cell growth. (B) Re‐expression of SMARCA4 or SMARCA2 was confirmed by western blotting. Ponceau S staining served as a loading control.