Role of BRCA1 Pathway in Regulating Oncogene-Induced Senescence

描述(申请人提供)：癌基因的定义是一种积极促进肿瘤形成的基因。矛盾的是，原代哺乳动物细胞中癌基因的激活，如RAS，通常会触发细胞衰老，导致永久性的生长停滞，从而提供一种抑制肿瘤发生的机制。我们广泛的长期目标是理解这种悖论背后的分子基础。衰老细胞的典型特征是DNA损伤的存在。RAS依赖的转化和衰老联系在一起的一个可能的原因可能是，损伤在建立衰老之前创造了一个支持转化的突变环境。BRCA1在DNA损伤的修复中起着关键作用，它的失活创造了一个驱动转化的突变环境。这项研究的理论基础是BRCA1在衰老诱导的细胞周期退出之前从染色质中释放出来。在转录水平上抑制BRCA1在DNA修复途径中的生理结合伙伴BRIP1会触发BRCA1染色质的解离。BRCA1的释放对衰老是必不可少的，因为异位BRIP1阻止BRCA1染色质的解离并阻止衰老。BRCA1染色质的解离也可能通过调节染色质结构来影响衰老。例如，BRCA1与依赖于ATP的染色质重构体BRG1相互作用。我们的初步研究表明，BRCA1和BRG1之间的相互作用在衰老过程中被破坏，这与染色质相关的BRG1水平增加相吻合。通过敲除BRCA1或异位表达BRG1来模仿这种效应会触发衰老。本研究的目的是确定BRCA1通路在调控癌基因诱导的衰老中的作用。我们的中心假设是原代细胞中由癌基因诱导的BRCA1染色质解离促进了绝大多数细胞的衰老。此外，它通过使BRCA1介导的DNA修复失活来创造一个突变环境，从而允许获得二次命中，并最终将少数细胞的命运转移到衰老旁路和转化。这一假说将在以下三个具体目标中得到验证：1)研究RAS诱导衰老过程中BRIP1抑制的机制；2)研究BRG1和BRCA1之间的相互作用改变促进衰老的机制；3)研究BRCA1介导的DNA修复反应，并确定DNA损伤是否促进衰老旁路和转化。提出的这项研究是创新的，因为它专注于一种新的细胞内在机制，它促进衰老，但同时也使细胞易于转化。提出的这项研究意义重大，因为它将调和癌基因诱导衰老领域的一个重大悖论。从我们的研究中获得的分子洞察力将揭示激活的癌基因如何促进衰老，同时使细胞易于转化，对癌症生物学具有广泛的意义。

DESCRIPTION (provided by applicant): The definition of an oncogene is a gene that actively promotes tumorigenesis. Paradoxically, activation of oncogenes, such as RAS, in primary mammalian cells usually triggers cellular senescence, which causes permanent growth arrest and thus provides a mechanism to suppress tumorigenesis. Our broad long-term goal is to understand the molecular basis underlying this paradox. Senescent cells are typically characterized by the presence of DNA damage. One possible reason for the linkage of RAS-dependent transformation and senescence might be that damage creates a mutagenic environment that supports transformation before establishing senescence. BRCA1 plays a critical role in the repair of DNA damage and its inactivation creates a mutagenic environment that drives transformation. The rationale for the proposed research is that BRCA1 is released from chromatin prior to senescence-induced cell cycle exit. Repression of BRIP1, a BRCA1 physiological binding partner in the DNA repair pathway, at transcriptional level triggers BRCA1 chromatin dissociation. BRCA1 release is essential for senescence, as ectopic BRIP1 prevents BRCA1 chromatin dissociation and blocks senescence. BRCA1 chromatin dissociation may also influence senescence through regulation of chromatin structure. For example, BRCA1 interacts with BRG1, an ATP dependent chromatin remodeler. Our preliminary studies show that the interaction between BRCA1 and BRG1 is disrupted during senescence and this coincides with an increased level of chromatin-associated BRG1. Mimicking this effect by knocking down BRCA1 or ectopically expressing BRG1 triggers senescence. The objective of this proposal is to determine the role of the BRCA1 pathway in regulation of oncogene-induced senescence. Our central hypothesis is that oncogene-induced BRCA1 chromatin dissociation in primary cells promotes senescence in the vast majority of cells. Further, it creates a mutagenic environment by inactivating BRCA1-mediated DNA repair, which allows for acquisition of secondary hits and ultimately diverts the fate of a minority of cells to senescence bypass and transformation. This hypothesis will be tested in the following three specific aims: 1) To investigate the mechanisms underlying BRIP1 repression during RAS-induced senescence; 2) To investigate the mechanism by which an altered interaction between BRG1 and BRCA1 promotes senescence; and 3) To investigate BRCA1-mediated DNA repair response in cells undergoing senescence, and determine whether DNA damage promotes senescence bypass and transformation. The research proposed is innovative because it focuses on a novel cell-intrinsic mechanism that promotes senescence but at the same time predisposes cells to transformation. The research proposed is significant because it will reconcile a major paradox in the oncogene-induced senescence field. The molecular insights gained from our studies will reveal how activated oncogenes promote senescence while predisposing cells to transformation with broad implications for cancer biology.