Genomic instability caused by defects in DNA repair and the DNA damage response is a common feature of many tumor cells. Epigenetic changes can also contribute to oncogenic transformation. However, the extent to which epigenetic changes can substitute for chromosomal instability is unclear, since highly malignant cancers are rarely diploid. Inactivation of SNF5 leads to aggressive tumors in children, and the median cancer onset following Snf5 loss in mouse models is remarkably short for inactivation of a single tumor suppressor. We hypothesized that the tumor suppressor activity of SNF5 arose not from a role in DNA repair or maintenance of genome integrity but rather from its role in the epigenetic control of gene expression.
Here we have demonstrated that loss of a gene involved in chromatin remodeling leads to aggressive cancers that lack other genomic aberrations. We show that Snf5 does not play a direct role in the DNA repair process or in the DNA damage response. Snf5 does not colocalize with DNA damage-induced γH2AX foci, its loss does not impair formation or resolution of γH2AX foci, and its loss does not confer sensitivity to DNA damaging agents that cause cross-links or DSBs. Furthermore, the DNA damage response is also intact, as indicated by normal phosphorylation of ATM, Chk1, and p53 and normal induction of p21, culminating in a G2 arrest following IR. Thus, we conclude that the mechanism of tumor suppression by SNF5 does not involve regulating the accessibility of damaged DNA to repair factors or initiating a proper DNA damage response.
Mammalian cells contain more than 20 SWI2/SNF2-related genes, and some of these, including INO80, SWR1, and RAD54, have been implicated in the execution of chromatin remodeling associated with DNA repair. Our results are in contrast to those in previous studies for which increased sensitivity to DNA damage in cells with conditionally inactivated Snf5 or overexpressed dominant-negative Brg1 was reported (33
). The reason for this discrepancy is not clear, although it may lie in the toxicity of these events. Both Snf5 inactivation and overexpression of dominant-negative BRG1 result in impaired proliferation after 5 days, making it difficult to interpret cellular responses to other stimuli. Once cellular viability begins to be impaired, cells may display reduced proliferation compared to normal cells when exposed to toxic agents (65
). We performed experiments early after Snf5 protein loss but before secondary effects upon growth had occurred (28
) in order to better distinguish sensitivity to DNA damaging agents from the lethal effects of Snf5 deletion. In addition to impaired proliferation, it is conceivable that overexpression of a dominant-negative version of the BRG1 subunit disrupts stoichiometry of the SWI/SNF complex and could cause off-target interactions that affect DNA repair. While we found no direct role for Snf5 in DNA repair nor any alteration of the DNA damage response up to 4 days after Snf5 deletion, it remains possible that some of the 1,400-plus genes whose transcription is affected by Snf5 loss (28
) may secondarily affect DNA repair at later time points. Additionally, while we are able to quantitatively remove the Snf5 protein as assessed by immunoblotting, it is conceivable that a small amount of protein remains bound to chromatin and obscures our ability to detect defects in DNA repair. We feel that the latter scenario is unlikely due to the absence of our ability to detect altered localization of Snf5 in response to IR, the absence of colocalization with γH2AX, the absence of abnormalities in numerous assays for DNA repair defects, and ultimately the fact that SNF5-deficient tumors are diploid and lack instability. While we have found no evidence of impaired DNA repair processes in Snf5-deficient cells or recurrent genomic amplifications/deletions in MRTs, it is important to note that we cannot rule out the possibility that pro-oncogenic point mutations exist in SNF5-deficient tumors. Doing this will ultimately require genome-wide single molecule DNA sequencing.
The intact DNA repair and damage response pathways in Snf5-deficient cells may underlie the surprising lack of genomic aberrations detected by chromosome counts of Snf5-deficient murine tumors and SNP arrays of human MRTs. Thus, we conclude that genomic instability does not account for the early onset and rapid development of SNF5-deficient tumors. Indeed, our data suggest that the epigenetic role of SNF5 in contributing to transcriptional regulation via nucleosome remodeling is the major, and perhaps sole, mechanism by which SNF5 acts as a tumor suppressor. Emerging evidence has implicated the chromatin remodeling activity of SWI/SNF in controlling the balance between cell proliferation and differentiation during development. Via the incorporation of lineage-restricted subunits, the SWI/SNF complex serves highly specific roles in fate determination in many tissues, processes known to go awry during transformation (2
). Loss of Snf5 disrupts developmental progression, alters the expression of numerous target genes, and has been implicated in known tumorigenic events that select for proliferation over differentiation, including specific upregulation of cyclin D1, p16INK4a
silencing, and derepression of E2F targets (6
). Furthermore, we and others have previously shown that Snf5 loss alters the expression of numerous target genes (22
). Thus, deregulation of transcriptional activities of the SWI/SNF complex due to SNF5 loss may be equivalent to multiple genetic changes, which undergo similar selective processes during tumorigenesis. In this model, SNF5 loss may result in “epigenetic instability” due to impaired nucleosome positioning or mobility. Since malpositioned nucleosomes presumably occur at numerous targets, oncogenesis and clonal selection may be dependent upon an individual cell being left with a specific combination of nucleosomes positioned both in silencing positions over tumor suppressor genes and in activating positions away from the promoters of genes that stimulate proliferation and survival. Oncogenic clonal selection, based upon the epigenetic state rather than the genetic state of a cell, may then explain the emergence of malignant cells from the otherwise widespread death caused by SNF5 loss in normal cells.
The epigenetic mechanisms acting in SNF5-deficient cancers likely are relevant to many other tumor types. A small percentage of numerous types of cancers, including acute myeloid leukemia, hepatocellular carcinoma, colorectal cancer, and others, do not exhibit detectable chromosomal or microsatellite instability (41
). Furthermore, while other pediatric solid tumors, such as Wilms' tumor, rhabdomyosarcoma, and Ewing's sarcoma, often have genomic anomalies, 56%, 35%, and 28% of these cancers, respectively, exhibit normal diploid genomes at the resolution of CGH (Progenetix CGH database [http://www.progenetix.de/~pgscripts/progenetix/
]). In addition, SNP arrays of acute lymphoblastic leukemias recently revealed a low instance of genomic aberrations in MLL-rearranged B-cell lineage acute lymphoblastic leukemia, with an average of one deletion per case. This is noteworthy, since MLL possesses histone methyltransferase activity, directly interacts with SNF5, and is recruited by SNF5 to cooperatively regulate tumor suppressor loci (31
), raising the possibility of a shared epigenetic mechanism between SNF5-mutant and MLL-mutant cancers. Collectively, these findings provide novel insight into the fundamental mechanisms required for oncogenic transformation and raise the intriguing possibility that epigenetic mechanisms can be sufficient to eliminate the need for genome instability during cancer formation.
Unlike genetic mutations, epigenetic changes are potentially reversible, and this may have important therapeutic relevance. Early attempts at developing drugs capable of altering the epigenetic state of cancer cells have met with some success, since the DNA methylation inhibitors decitabine and 5-azacytidine have both been FDA approved for the treatment of myelodysplastic syndrome and the histone deacetylase inhibitor vorinostat has been FDA approved for cutaneous T-cell lymphoma. Since the large majority of cancers that arise following SNF5 loss lack genomic aberrations, these tumors may constitute a useful model both which to investigate the mechanisms by which epigenetic changes contribute to oncogenesis and also with which to test therapeutic interventions aimed at reversing the epigenetic state of cancer.