Pathways of oncogene-induced senescence at DNA replication forks

项目摘要/摘要 癌基因诱导的衰老(OIS)是由癌基因的异常激活引发的，如突变形式 RAS是抑制癌前病变进展的关键屏障。OIS伴随着 DNA复制压力增加和DNA损伤反应(DDR)激活；开创性研究表明 复制相关的DNA损伤是建立OIS的先决条件，在OIS中，细胞必须活跃 复制导致复制应激增加，从而触发DDR以响应癌基因诱导的DNA高表达 复制。相反，OIS的旁路允许受损细胞的增殖和致癌转化。 基因组的不稳定性。虽然这些想法强调OIS是由DNA损伤造成的抗癌屏障 检查点，致癌刺激的确切性质，最终导致异常DNA复制和激活 停滞不前的复制分叉的DDR仍然难以捉摸。此外，是否存在明显的细胞信号 将上游致癌信号传递到复制分叉以导致DNA复制分叉不稳定的级联 尚未被发现。在这个应用中，我们建议解释癌基因诱导的机制 复制压力对DNA复制分叉的完整性。具体地说，我们在分叉中推理出永恒(TIM) 保护复合体，是复制分叉进展和复制所必需的复制体的基本支架 检查点激活，是OIS的关键决定因素。我们的初步数据表明，RAS诱导的OIS 激活与TIM的下调有关，该下调与一种新的翻译后修饰有关，即 依赖于PARP1的多聚ADP-TIM核糖化及其蛋白降解。我们假设，不能 通过抑制TIM活性来保护停滞的叉子是一种癌基因诱导的机制，它有助于 复制压力的积累和DNA分叉的不稳定性。我们将以HRASG12V作为模型癌基因来诱导 未转化的人成纤维细胞中的OIS。在第一个目标中，我们将确定HRASG12V诱导的机制 通过TIM下调建立OIS，并确定TIM击倒在触发DNA复制中的作用 叉子不稳和衰老。我还将调查TIM过度表达是否克服了DNA复制 叉子不稳定，从而导致OIS的发病。各种分子和细胞技术将被使用， 包括DNA梳理分析，DNA复制叉切，以评估TIM耗竭细胞的能力 以防止因癌基因激活而导致的分叉降解。在第二个目标中，我们将确定机制 OIS期间TIM下调的研究，重点是多聚ADP核糖化和蛋白水解酶的特征 由HRASG12V驱动的信号级联所触发的TIM降解。在第三个目标中，我们将阐明 在Kras驱动的前列腺癌器官模型中TIM在促进肿瘤发生中的作用 早期致癌事件的主要参与者，以打破OIS障碍并促进在 RAS激活。总之，我们希望揭示OIS发病的新机制，最终定义 TIM作为肿瘤治疗的靶点，诱导癌细胞衰老。

PROJECT SUMMARY/ABSTRACT Oncogene-induced senescence (OIS) is triggered by aberrant activation of oncogenes such as the mutant form of RAS and acts as a key barrier to inhibit the progression of preneoplastic lesions. OIS is accompanied by elevated DNA replication stress and activation of the DNA damage response (DDR); seminal studies have shown that replication-associated DNA damage is a prerequisite for establishing OIS, where cells must be actively replicating leading to a rise in replication stress that triggers DDR in response to oncogene-induced DNA hyper- replication. Conversely, bypass of OIS allows the proliferation and oncogenic transformation of damaged cells with genome instability. While these ideas underscore OIS as an anti-cancer barrier imposed by DNA damage checkpoints, the precise nature of oncogenic stimuli that culminate in aberrant DNA replication and activation of the DDR at stalled replication forks remains elusive. In addition, whether there exists distinct cellular signaling cascade that relays upstream oncogenic signaling to a replication fork to cause DNA replication fork instability is yet to be discovered. In this application, we propose to explicate the mechanisms that link oncogene-induced replication stress to DNA replication fork integrity. Specifically, we reason that TIMELESS (TIM) in the fork protection complex, an essential scaffold of the replisome necessary for replication fork progression and checkpoint activation, is a key determinant of OIS. Our preliminary data indicate that OIS induced by RAS activation is associated with downregulation of TIM that is linked to a novel posttranslational modification, i.e. PARP1-dependent polyADP-ribosylation, of TIM and its proteolytic degradation. We hypothesize that failure to protect stalled forks via suppression of TIM activity is an oncogene-induced mechanism that contributes to the accumulation of replication stress and DNA fork instability. We will use HRASG12V as a model oncogene to induce OIS in non-transformed human fibroblasts. In the first aim, we will identify the mechanism of HRASG12V-induced OIS established by TIM downregulation and determine the role of TIM knockdown in triggering DNA replication fork instability and senescence. I will also investigate whether TIM overexpression overcomes DNA replication fork instability, and thus the onset of OIS. A variety of molecular and cellular techniques will be employed, including DNA combing analysis of DNA replication fork resection, to evaluate the capacity of TIM-depleted cells to prevent fork degradation in response to oncogene activation. In the second aim, we will identify the mechanism of TIM downregulation during OIS, focusing on characterizing the polyADP-ribosylation and proteolytic degradation of TIM triggered by the HRASG12V-driven signaling cascade. In the third aim, we will elucidate the role of TIM in promoting tumorigenesis in the Kras-driven prostate cancer organoid model to define TIM as a major player of early oncogenic events to breach the OIS barrier and promote transformation in the context of RAS activation. Together, we expect to reveal a new mechanism underlying the onset of OIS, ultimately defining TIM as a target of cancer therapy to induce senescence of cancer cells.