Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer.
Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer.
The active DNA-dependent ATPase A domain (ADAAD), a member of the SWI2/SNF2 family, has been shown to bind DNA in a structure-specific manner, recognizing DNA molecules possessing double-stranded to single-stranded transition regions leading to ATP hydrolysis. Extending these studies we have delineated the structural requirements of the DNA effector for ADAAD and have shown that the single-stranded and double-stranded regions both contribute to binding affinity while the double-stranded region additionally plays a role in determining the rate of ATP hydrolysis. We have also investigated the mechanism of interaction of DNA and ATP with ADAAD and shown that each can interact independently with ADAAD in the absence of the other. Furthermore, the protein can bind to dsDNA as well as ssDNA molecules. However, the conformation change induced by the ssDNA is different from the conformational change induced by stem-loop DNA (slDNA), thereby providing an explanation for the observed ATP hydrolysis only in the presence of the double-stranded:single-stranded transition (i.e. slDNA).
Many members of the SWI2/SNF2 family of adenosine triphosphatases participate in the assembly/disassembly of multiprotein complexes involved in the DNA metabolic processes of transcription, recombination, repair, and chromatin remodeling. The DNA molecule serves as an essential effector or catalyst for most of the members of this particular class of proteins, and the structure of the DNA may be more important than the nucleotide sequence. Inspection of the DNA structure at sites where multiprotein complexes are assembled/disassembled for these various DNA metabolic processes reveals the presence of a common element: a double-stranded to single-stranded transition region. We now show that this DNA element is crucial for the ATP hydrolytic function of an SWI2/SNF2 family member: DNA-dependent ATPase A. We further demonstrate that a domain containing the seven helicase-related motifs that are common to the SWI2/SNF2 family of proteins mediates the interaction with the DNA, yielding specific DNA structural recognition. This study forms a primary step toward understanding the physico-biochemical nature of the interaction between a particular class of DNA-dependent ATPase and their DNA effectors. Furthermore, this study provides a foundation for development of mechanisms to specifically target this class of DNAdependent ATPases.DNA recognition and the ensuing ATP hydrolysis by DNAdependent ATPases are common denominators of a number of different macromolecular assembly/disassembly processes that are required for DNA metabolic events such as chromatin remodeling, replication, repair, recombination, and transcription. There are a variety of types of known nucleic acid-modifying enzymes that use the energy released by ATP hydrolysis to produce changes in a nucleic acid substrate (i.e. helicases, nucleases, topoisomerases, ligases, and recombinases). However, there are a few nucleic acid-dependent ATPases where the nucleic acid is not obviously modified and consequently does not appear to be a substrate for the enzyme. In these cases, the nucleic acid is generally regarded as an effector of the enzymatic activity. DNA-dependent ATPase A hydrolyzes ATP only in the presence of a DNA effector, without apparent modification of the effector. Originally isolated from calf thymus tissue, ATPase A is a 105-kDa protein that undergoes proteolysis to yield two smaller polypeptides with estimated molecular masses of 68 and 83 kDa (1). All three polypeptides possess ATPase activity that is manifested only in the presence of DNA. Initial studies with the 68-kDa polypeptide indicated that the protein is maximally active in the presence of DNA effectors possessing primer-template junctions (i.e. doublestranded to single-stranded transition). This effector preference is reminiscent of the gp44⅐62 and gp45 protein complex from T4 bacteriophage (2, 3). The gp44⅐62 protein complex hydrolyzes ATP in the presence of primer-template DNA, with the energy release being coupled to the loading of a gp45 sliding clamp complex onto DNA, thereby incre...
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