Pathways of oncogene-induced senescence at DNA replication forks

项目概要/摘要 癌基因诱导的衰老（OIS）是由癌基因的异常激活触发的，如突变形式 的RAS，并作为一个关键的障碍，以抑制肿瘤前病变的进展。OIS伴随着 升高的DNA复制应激和DNA损伤反应（DDR）的激活;开创性研究表明， 复制相关的DNA损伤是建立OIS的先决条件，细胞必须积极地 复制导致复制应激的上升，从而触发DDR，以响应癌基因诱导的DNA超增殖， 复制的相反，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 延迟的复制分叉处的DDR仍然难以捉摸。此外，是否存在不同的细胞信号传导， 将上游致癌信号传递到复制叉以引起DNA复制叉不稳定性的级联反应 还有待发现在这个应用中，我们建议阐明连接癌基因诱导的细胞凋亡的机制。 复制应激对DNA复制叉完整性的影响。具体地说，我们的理由是，TIMELESS（TIM）在分叉中 保护复合物，复制叉进展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，TIM与新的翻译后修饰有关，即 TIM及其蛋白水解降解的PARP 1依赖性聚ADP-核糖基化。我们假设， 通过抑制TIM活性来保护停滞的分叉是一种癌基因诱导的机制， 复制应激和DNA叉不稳定性的积累。我们将使用HRASG 12 V作为模型癌基因来诱导 非转化人成纤维细胞中的OIS。在第一个目标中，我们将确定HRASG 12 V诱导的HRASG 12 V的机制。 通过TIM下调建立OIS，并确定TIM敲低在触发DNA复制中的作用 分叉不稳定和衰老。我还将研究TIM过表达是否能克服DNA复制 分叉不稳定，从而引发OIS。将采用各种分子和细胞技术， 包括DNA复制叉切除的DNA梳理分析，以评估TIM缺失细胞的能力 以防止癌基因激活引起的叉降解。在第二个目标中，我们将确定机制 在OIS期间TIM下调，重点是表征多聚ADP-核糖基化和蛋白水解， 由HRASG 12 V驱动的信号级联触发的TIM降解。在第三个目标中，我们将阐明 TIM在Kras驱动的前列腺癌类器官模型中促进肿瘤发生的作用，以将TIM定义为 早期致癌事件的主要参与者，以突破OIS屏障并促进转化， RAS激活。总之，我们希望揭示OIS发病的新机制，最终定义 TIM作为癌症治疗的靶点以诱导癌细胞衰老。

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.