Mechanism of BRG1 silencing in primary cancers

Abstract

BRG1 (SMARCA4) is a documented tumor suppressor and a key subunit of the SWI/SNF chromatin remodeling complex that is silenced in many cancer types. Studies have shown that BRG1 is mutated in cancer-derived cell lines, which led to the assertion that BRG1 is also mutated in primary human tumors. However, the sequencing of BRG1-deficient tumors has revealed a paucity of mutations; hence, the cause of BRG1 silencing in tumors remains an enigma. We conducted immunohistochemistry (IHC) on a number of tumor microarrays to characterize the frequency of BRG1 loss in different tumor types. We also analyzed BRG1-deficient tumors by sequencing the genomic DNA and the mRNA. We then tested if BRG1 expression could be induced in BRG1-negative cell lines (i.e., that lack mutations in BRG1) after the application of several different epigenetic agents, including drugs that inhibit the AKT pathway. We found that a subset of BRG1-negative cell lines also demonstrated aberrant splicing of BRG1, and in at least 30% of BRG1-deficient tumors, BRG1 expression appeared to be suppressed due to aberrant BRG1 splicing.As the majority of BRG1-deficient tumors lack mutations or splicing defects that could drive BRG1 loss of expression, this suggests that other mechanisms underlie BRG1 silencing. To this end, we analyzed 3 BRG1-deficient nonmutated cancer cell lines and found that BRG1 was inducible in these cell lines upon inhibition of the AKT pathway. We show that the loss of BRG1 is associated with the loss of E-cadherin and up-regulation of Vimentin in primary tumors, which explains why BRG1 loss is associated with a poor prognosis in multiple tumor types.

BRG1 and BRM loss selectively impacts RB and P53, respectively: BRG1 and BRM have differential functions in vivo

“…Moreover, in vitro overexpression experiments are problematic in addressing this question because the strong viral promoters often used yield such high levels that the ectopic expression of BRG1 or BRM activates both BRG1- and BRM-dependent SWI/SNF complexes [15]. Further, shRNA knockdown experiments do not completely deplete BRG1 and BRM in BRG1/BRM- positive cell lines, and low levels of BRG1/BRM can still have an impact on cell phenotypes [23]. It has therefore been difficult to differentiate the separate functions of BRG1 versus BRM.…”

“…However, there are a number of drugs used clinically, such as TKIs and CDK inhibitors that inhibit growth in part by activating RB1 [50, 57]. In our previous work, we showed that BRG1 could be silenced by aberrant splicing (irreversibly silenced) or translationally blocked (reversibly silenced) [23], as in the Sum159 and DT13 cell lines. Hence, the treatment of these cancer cells, where BRG1 is reversibly silenced, with TKIs (e.g., Sorafenib and PPP) appears to induce BRG1 and inhibit growth.…”

“…However, unlike TP53 , which is principally inactivated by mutations, BRM is almost never mutated (<2%) but, rather, it is epigenetically silenced [22]. Similarly, the frequency of BRG1 mutations is also relatively low (<2- 5%) in most tumors [22], and our recent work has shown that BRG1 can be silenced by either aberrant splicing or translational blocking mechanisms regulated by the AKT pathway [23]. Interestingly, ARID1A , which is usually inactivated by mutation, does indeed show an inverse correlation with TP53 mutations in a number of tumor types [24-26].…”

Roles of pRB in the Regulation of Nucleosome and Chromatin Structures

“…In this context, the involvement of pRB directs antitumorigenesis via a conformational change in the local promoter region with or without epigenetic marks. In many types of human cancer cells, the levels of pRB and pRB binding nucleosome/chromatin-related proteins that act cooperatively with pRB, such as HDACs [42], PML [98], and BRG1 [108], are decreased. Furthermore, loss of Suv4-20h1 in breast cancer cells was reported [109].…”

Two BRM promoter polymorphisms predict poor survival in patients with hepatocellular carcinoma

“…In primary tumors, BRG1 appears to be silenced via aberrant splicing and/or microRNA expression rather than mutations or alterations in methylation . Similarly, BRM is primarily silenced via epigenetic mechanisms that involve BRM promoter polymorphism .…”