Courtney G. Sansam

Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry.

Bcl‐xL and Bcl‐2 inhibit both apoptosis and proliferation. In investigating the relationship between these two functions of Bcl‐xL and Bcl‐2, an analysis of 24 Bcl‐xL and Bcl‐2 mutant alleles, including substitutions at residue Y28 previously reported to selectively abolish the cell cycle activity, showed that cell cycle delay and anti‐apoptosis co‐segregated in all cases. In determining whether Bcl‐2 and Bcl‐xL act in G0 or G1, forward scatter and pyronin Y fluorescence measurements indicated that Bcl‐2 and Bcl‐xL cells arrested more effectively in G0 than controls, and were delayed in G0–G1 transition. The cell cycle effects of Bcl‐2 and Bcl‐xL were reversed by Bad, a molecule that counters the survival function of Bcl‐2 and Bcl‐xL. When control and Bcl‐xL cells of equivalent size and pyronin Y fluorescence were compared, the kinetics of cell cycle entry were similar, demonstrating that the ability of Bcl‐xL and Bcl‐2 cells to enhance G0 arrest contributes significantly to cell cycle delay. Our data suggest that cell cycle effects and increased survival both result from intrinsic functions of Bcl‐2 and Bcl‐xL.

Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog-Gli pathway

Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli–activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.

Oncogenesis Caused by Loss of the SNF5 Tumor Suppressor Is Dependent on Activity of BRG1, the ATPase of the SWI/SNF Chromatin Remodeling Complex

Alterations in chromatin play an important role in oncogenic transformation, although the underlying mechanisms are often poorly understood. The SWI/SNF complex contributes to epigenetic regulation by using the energy of ATP hydrolysis to remodel chromatin and thus regulate transcription of target genes. SNF5, a core subunit of the SWI/SNF complex, is a potent tumor suppressor that is specifically inactivated in several types of human cancer. However, the mechanism by which SNF5 mutation leads to cancer and the role of SNF5 within the SWI/SNF complex remain largely unknown. It has been hypothesized that oncogenesis in the absence of SNF5 occurs due to a loss of function of the SWI/SNF complex. Here, we show, however, distinct effects for inactivation of Snf5 and the ATPase subunit Brg1 in primary cells. Further, using both human cell lines and mouse models, we show that cancer formation in the absence of SNF5 does not result from SWI/SNF inactivation but rather that oncogenesis is dependent on continued presence of BRG1. Collectively, our results show that cancer formation in the absence of SNF5 is dependent on the activity of the residual BRG1-containing SWI/SNF complex. These findings suggest that, much like the concept of oncogene addiction, targeted inhibition of SWI/SNF ATPase activity may be an effective therapeutic approach for aggressive SNF5-deficient human tumors.

Swi/Snf chromatin remodeling/tumor suppressor complex establishes nucleosome occupancy at target promoters

Precise nucleosome-positioning patterns at promoters are thought to be crucial for faithful transcriptional regulation. However, the mechanisms by which these patterns are established, are dynamically maintained, and subsequently contribute to transcriptional control are poorly understood. The switch/sucrose non-fermentable chromatin remodeling complex, also known as the Brg1 associated factors complex, is a master developmental regulator and tumor suppressor capable of mobilizing nucleosomes in biochemical assays. However, its role in establishing the nucleosome landscape in vivo is unclear. Here we have inactivated Snf5 and Brg1, core subunits of the mammalian Swi/Snf complex, to evaluate their effects on chromatin structure and transcription levels genomewide. We find that inactivation of either subunit leads to disruptions of specific nucleosome patterning combined with a loss of overall nucleosome occupancy at a large number of promoters, regardless of their association with CpG islands. These rearrangements are accompanied by gene expression changes that promote cell proliferation. Collectively, these findings define a direct relationship between chromatin-remodeling complexes, chromatin structure, and transcriptional regulation.

Loss of the Epigenetic Tumor Suppressor SNF5 Leads to Cancer without Genomic Instability

ABSTRACT There is a growing appreciation of the role that epigenetic alterations can play in oncogenesis. However, given the large number of genetic anomalies present in most cancers, it has been difficult to evaluate the extent to which epigenetic changes contribute to cancer. SNF5 (INI1/SMARCB1/BAF47) is a tumor suppressor that regulates the epigenome as a core member of the SWI/SNF chromatin remodeling complex. While the SWI/SNF complex displays potent tumor suppressor activity, it is unknown whether this activity is exerted genetically via maintenance of genome integrity or epigenetically via transcriptional regulation. Here we show that Snf5-deficient primary cells do not show altered sensitivity to DNA damaging agents, defects in γ-H2AX induction, or an abrogated DNA damage checkpoint. Further, the aggressive malignancies that arise following SNF5 loss are diploid and genomically stable. Remarkably, we demonstrate that most human SNF5-deficient cancers lack genomic amplifications/deletions and, aside from SNF5 loss, are indistinguishable from normal cells on single-nucleotide polymorphism arrays. Finally, we show that epigenetically based changes in transcription that occur following SNF5 loss correlate with the tumor phenotype. Collectively, our results provide novel insight into the mechanisms of oncogenesis by demonstrating that disruption of a chromatin remodeling complex can largely, if not completely, substitute for genomic instability in the genesis of aggressive cancer.

SWI/SNF Deficiency Results in Aberrant Chromatin Organization, Mitotic Failure, and Diminished Proliferative Capacity

Switch (SWI)/sucrose nonfermentable (SNF) is an evolutionarily conserved complex with ATPase function, capable of regulating nucleosome position to alter transcriptional programs within the cell. It is known that the SWI/SNF complex is responsible for regulation of many genes involved in cell cycle control and proliferation, and it has recently been implicated in cancer development. The ATPase action of SWI/SNF is conferred through either the brahma-related gene 1 (Brg1) or brahma (Brm) subunit of the complex, and it is of central importance to the modification of nucleosome position. In this study, the role of the Brg1 and Brm subunits were examined as they relate to chromatin structure and organization. Deletion of the Brg1 ATPase results in dissolution of pericentromeric heterochromatin domains and a redistribution of histone modifications associated with these structures. This effect was highly specific to Brg1 and is not reproduced by the loss of Brm or SNF5/BAF47/INI1. Brg1 deficiency is associated with the appearance of micronuclei and aberrant mitoses that are a by-product of dissociated chromatin structure. Thus, Brg1 plays a critical role in maintaining chromatin structural integrity.

Epigenetics and cancer: altered chromatin remodeling via Snf5 loss leads to aberrant cell cycle regulation.

The term ‘epigenetics’ refers to heritable changes in gene function that occur in theabsence of any change in DNA sequence. Perturbations of epigenetic gene regulation mayplay a critical role in the genesis of most, if not all, cancers. These alterations include changesin covalent modifications of DNA and histones as well as non-covalent changes in nucleosomepositioning. Covalent epigenetic modifications have been the main focus of cancerinvestigation, perhaps because they are more readily assayed and understood than noncovalentmodifications. Recently, evidence has emerged demonstrating that perturbation ofcomplexes that remodel the structure of chromatin by mobilizing nucleosomes may have a keyrole in tumor suppression and oncogenic transformation. For example, Snf5(Ini1/Baf47/Smarcb1), a core component of the Swi/Snf ATPase chromatin remodelingcomplex, is a potent tumor suppressor that is specifically inactivated in lethal childhoodcancers. Notably, these cancers may serve as a paradigm for epigenetic cancers as, despitetheir extremely aggressive nature, the majority have an entirely normal karyotype with onlymicrodeletions at the Snf5 locus. Recent investigations have shed light upon the mechanisticbasis of Snf5 function by demonstrating that Snf5 and the Swi/Snf complex regulate the cellcycle and cooperate with p53 to prevent oncogenic transformation.

G0 function of BCL2 and BCL-xL requires BAX, BAK, and p27 phosphorylation by Mirk, revealing a novel role of BAX and BAK in quiescence regulation.

BCL2 and BCL-xL facilitate G0 quiescence by decreasing RNA content and cell size and up-regulating p27 protein, but the precise mechanism is not understood. We investigated the relationship between cell cycle regulation and the anti-apoptosis function of BCL2 and BCL-xL. Neither caspase inhibition nor abrogation of mitochondria-dependent apoptosis by BAX and BAK deletion fully recapitulated the G0 effects of BCL2 or BCL-xL, suggesting that mechanisms in addition to anti-apoptosis are involved in the cell cycle arrest function of BCL2 or BCL-xL. We found that BCL2 and BCL-xL expression in bax-/- bak-/- cells did not confer cell cycle effects, consistent with the G0 function of BCL2 and BCL-xL being mediated through BAX or BAK. Stabilization of p27 in G0 in BCL2 or BCL-xL cells was due to phosphorylation of p27 at Ser10 by the kinase Mirk. In bax-/- bak-/- cells, total p27 and p27 phosphorylated at Ser10 were elevated. Re-expression of BAX in bax-/- bak-/- cells and silencing of BAX and BAK in wild type cells confirmed that endogenous BAX and BAK modulated p27. These data revealed a novel role for BAX and BAK in the regulation of G0 quiescence.

TCR-dependent transformation of mature memory phenotype T cells in mice

A fundamental goal in cancer research is the identification of the cell types and signaling pathways capable of initiating and sustaining tumor growth, as this has the potential to reveal therapeutic targets. Stem and progenitor cells have been implicated in the genesis of select lymphoid malignancies. However, the identity of the cells in which mature lymphoid neoplasms are initiated remains unclear. Here, we investigate the origin of peripheral T cell lymphomas using mice in which Snf5, a chromatin remodelling-complex subunit with tumor suppressor activity, could be conditionally inactivated in developing T cells. In this model of mature peripheral T cell lymphomas, the cell of origin was a mature CD44hiCD122loCD8⁺ T cell that resembled a subset of memory cells that has capacity for self-renewal and robust expansion, features shared with stem cells. Further analysis showed that Snf5 loss led to activation of a Myc-driven signaling network and stem cell transcriptional program. Finally, lymphomagenesis and lymphoma proliferation depended upon TCR signaling, establishing what we believe to be a new paradigm for lymphoid malignancy growth. These findings suggest that the self-renewal and robust proliferative capacities of memory T cells are associated with vulnerability to oncogenic transformation. Our findings further suggest that agents that impinge upon TCR signaling may represent an effective therapeutic modality for this class of lethal human cancers.

Epigenetic inactivation of the tumor suppressor BIN1 drives proliferation of SNF5-deficient tumors

Emerging evidence demonstrates that subunits of the SWI/SNF chromatin remodeling complex are specifically mutated at high frequency in a variety of human cancer types. SNF5 (SMARCB1/INI1/BAF47), a core subunit of the SWI/SNF complex, is inactivated in the vast majority of rhabdoid tumors (RT), an aggressive type of pediatric cancer. SNF5-deficient cancers are diploid and genomically stable, suggesting that epigenetically based changes in transcription are key drivers of tumor formation caused by SNF5 loss. However, there is limited understanding of the target genes that drive cancer formation following SNF5 loss. Here we performed comparative expression analyses upon three independent SNF5-deficient cancer data sets from both human and mouse and identify downregulation of the BIN1 tumor suppressor as a conserved event in primary SNF5-deficient cancers. We show that SNF5 recruits the SWI/SNF complex to the BIN1 promoter, and that the marked reduction of BIN1 expression in RT correlates with decreased SWI/SNF occupancy. Functionally, we demonstrate that re-expression of BIN1 specifically compromises the proliferation of SNF5-deficient RT cell lines. Identification of BIN1 as a SNF5 target gene reveals a novel tumor suppressive regulatory mechanism whose disruption can drive cancer formation.