SMARCB1 protein and mRNA loss is not caused by promoter and histone hypermethylation in epithelioid sarcoma

Abstract

About 10% of epithelioid sarcomas have biallelic mutation of the SMARCB1 (SWI/SNF-related, matrixassociated, actin-dependent regulator of chromatin, subfamily b, member 1) gene resulting in a lack of this nuclear protein. It has been suggested that SMARCB1 may be silenced by epigenetic changes in the remaining 90% of tumors. Thus, we hypothesized that the promoter of SMARCB1 is hypermethylated. We also examined SMARCB1 mRNA level to determine if a post-translational change was possible. Thirty-six cases of epithelioid sarcomas were studied. Immunohistochemistry and mutation analysis of the SMARCB1 gene were performed to select appropriate cases. Methylation status was assessed by methylation-specific PCR. Laser capture microdissection of tumor cells followed by real-time PCR was applied to examine the expression of SMARCB1 mRNA. Of 36 epithelioid sarcomas, 31 (86%) displayed a lack of SMARCB1 nuclear protein. In all, 4 (13%) of 31 SMARCB1-negative cases harbored biallelic deletion while 9 (33%) cases showed single-allelic deletion. One (4%) frameshift deletion of exon 3 and one point mutation of exon 7 were also found. In 16 (59%) cases, both alleles were intact. Altogether, 25/31 (81%) SMARCB1-negative cases had at least one intact allele. None of these cases demonstrated promoter hypermethylation. Low levels of SMARCB1 mRNA were found in all cases with tumor tissue extracted RNA (because of the minimal normal cell contamination) but no mRNA could be detected in laser dissected cases (containing only tumor cells). Enhancer of zeste homolog 2 (EZH2) overexpression was not characteristic of epithelioid sarcoma. Thus, loss of SMARCB1 expression in epithelioid sarcoma is caused neither by DNA hypermethylation nor by post-translational modifications. Most likely it is the microRNA destruction of SMARCB1 mRNA but further investigations are needed to elucidate this issue. Keywords: epithelioid sarcoma; histone methylation; laser capture microdissection; methylation-specific PCR; promoter hypermethylation; SMARCB1The SMARCB1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1; also known as INI1, BAF47 and hSNF5) gene, located on chromosomal position 22q11.23, encodes a core subunit of the SWI/SNF ATP-dependent chromatin remodeling complex. 1 Such observations show these complexes regulate gene expression, at least in part, by inducing a nucleosome conformation that is more approachable to the transcriptional machinery. 2 Recently, inactivation of both alleles of the SMARCB1 gene was identified in the majority of malignant rhabdoid tumors, suggesting that loss-of-function mutations of the gene contributed to the oncogenesis of this type of tumor.

The chromatin remodelling component SMARCB1/INI1 influences the metastatic behavior of colorectal cancer through a gene signature mapping to chromosome 22

“…The molecular mechanisms underlying SMARCB1/INI1 protein inactivation in CRC were not explored in the present study; however, in agreement with recent data, we ruled out hypermethylation of the SMARCB1/INI1 promoter region in our CRC cohort (our unpublished data) [17,21,22]. A recent comprehensive genome-wide analysis on 276 CRCs has identified SMARCB1/INI1 mutations in less than 1% of cases [21].…”

Secondary EWSR1 gene abnormalities in SMARCB1‐deficient tumors with 22q11‐12 regional deletions: Potential pitfalls in interpreting EWSR1 FISH results

“…As no FISH probes are available for SMARCB1 , a number of labs and studies use as a surrogate the anti- BCR telomeric probe, which targets a 645-kb long region (23,986–24,631 kb genomic position, spanning the SMARCB1 gene, 24,129–24,176 kb) (Papp et al, 2013). However, this commercial probe covers a bigger target region (fourfold larger than our specific custom probe, 148 kb), including the centromeric end of the SMARCB1 gene.…”

Frequent co‐inactivation of the SWI/SNF subunits SMARCB1, SMARCA2 and PBRM1 in malignant rhabdoid tumours

“…In mammals, the SWI/SNF complex is a polymorphic assembly of at least 13 subunits encoded by 26 genes, mainly including SMARCB1, SMARCA4 (BRG1), SMARCA2 (BRM), ARID1A (BAF250A), and PBRM1 (BAF180), which are critical for growth and cancer development . Frequent inactivation of these genes has been reported in various tumours, including renal, bladder, prostate, ovarian, endometrioid, hepatocellular, gastric, breast, lung, pancreatic, colon and soft tissue malignancy in recent studies . Although SMARCB1 has been extensively reported in the pathogenesis of MRTs, recent studies also suggest that inactivation or absence of one subunit could be compensated for by other subunits of the SWI/SNF complex, which implies the potential involvement of other subunits in MRTs.…”