Fhit as modulator of Chk1 termination and response to DNA-damaging agents

描述（由申请人提供）：DNA损伤反应（DDR）已被认为是肿瘤发生的屏障，使得肿瘤前病变显示出激活的DDR检查点，从而阻碍细胞增殖并诱导细胞衰老和/或凋亡。矛盾的是，DNA 损伤检查点被认为对癌细胞的生存很重要，因为这些细胞产生的 DNA 损伤程度（无论是内源性的还是通过 DNA 靶向治疗），以及需要修复才能从损伤中恢复。最近有人提出，向 S/G2 检查点发出信号的 Chk1 激酶降解机制的缺陷可能会导致癌细胞丧失对某些抗癌药物的敏感性。有趣的是，FHIT（一种复制应激靶标）在多种类型的癌症中经常发生改变，这些改变导致 Fhit 蛋白表达丧失。此外，Fhit 在癌细胞中的重新表达已被证明可以调节 DDR 检查点并使癌细胞对 DNA 损伤敏感。我们最近的研究结果表明，Fhit 缺陷的癌细胞在暴露于 DNA 复制应激时表现出异常的 DNA 损伤检查点反应和延长的 S 期 Chk1 激活。这些观察结果表明，在具有持续复制叉阻断和/或广泛 DNA 损伤的细胞中，Fhit 作为信号传导 Chk1 失活的参与者发挥着以前未知的作用。基于这些发现，我们假设 Fhit 定向的 Chk1 终止使癌细胞对 DNA 靶向抗癌药物敏感，并且肿瘤中 Fhit 表达的丧失是细胞对基因毒性药物无反应且对 DNA 损伤不敏感的特征。 Fhit 的这种肿瘤抑制功能可消除具有累积 DNA 损伤的癌细胞，因此可能有助于 DDR 肿瘤发生屏障。该项目的目标是测试这些假设，定义 Fhit 定向 Chk1 终止的机制，并评估这些发现的体内和临床相关性。为此，我们将使用 H1299 肺癌和 MKN74 胃癌细胞系检查 Fhit 调节 Chk1 信号传导的机制。我们假设 Fhit 促进 pChk1 的泛素化和降解。接下来，我们将在一组癌细胞系中确认我们的机制发现，并确定癌细胞中 Fhit 缺陷和 Chk1 信号失活失败的生物学后果。最后，我们将检查 300 个三阴性乳腺癌的 TMA，以了解 Fhit 表达的丧失和 Chk1 通路的激活。该 TMA 将与无病生存等临床特征相关，从而将 Fhit 表达缺失与临床结果联系起来。获得的结果将有助于深入了解 Fhit 作为肿瘤抑制因子的功能、Chk1 失活失败的机制以及有助于癌细胞进展和治疗存活的缺陷。重要的是，这些结果还将有助于确定测试 Chk1 和 Parp1 抑制剂与化疗药物联合治疗某些类型癌症的临床前研究的基本原理，以及确定将从此类疗法中受益的患者的标准。 公共健康相关性：所有细胞都有 DNA 修复系统和“检查点”，以确保细胞在受到过多损伤时无法生存，但癌细胞具有过度激活的“检查点”，即使它们没有修复所有 DNA 损伤，它们也能生存和生长。 Fhit 肿瘤抑制蛋白表达的缺失增强了癌细胞在广泛的 DNA 损伤（甚至是化疗造成的损伤）中生存的能力。该项目将定义 Fhit 调节“检查点”的机制，从而有助于理解癌细胞中异常的“检查点”。

DESCRIPTION (provided by applicant): The DNA damage response (DDR) has been proposed to be a barrier to tumorigenesis, such that preneoplastic lesions show activated DDR checkpoints that impede cell proliferation and induce cell senescence and/or apoptosis. Paradoxically, DNA damage checkpoints have been proposed to be important for cancer cell survival due to the extent of DNA damage incurred by these cells, either endogenously or by DNA-targeting therapy, and the need for repair to recover from damage. It was recently proposed that defects in degradation machinery for the Chk1 kinase that signals S/G2 checkpoints, could result in loss of cancer cell sensitivity to certain anticancer drugs. Interestingly, FHIT, a replicative stress target, is frequently altered in several types of cancer, alterations resulting in the loss of Fhit protein expression. Furthermore, re-expression of Fhit in cancer cells has been shown to modulate the DDR checkpoints and sensitize cancer cells to DNA damage. Our recent findings suggest that Fhit-deficient cancer cells exhibit an aberrant DNA damage checkpoint response and prolonged S-phase Chk1 activation while exposed to DNA replication stress. These observations imply a previously unknown role of Fhit as a participant in signaling Chk1 deactivation in cells with sustained replication fork block and/or extensive DNA damage. Based on these findings, we hypothesize that Fhit-directed Chk1 termination sensitizes cancer cells to DNA-targeting anticancer drugs, and that loss of Fhit expression in tumors is characteristic of cells that are unresponsive to genotoxic agents and insensitive to DNA damage. This proposed tumor suppressive function of Fhit serves to eliminate cancer cells with accumulated DNA damage, and therefore would likely contribute to the DDR tumorigenesis barrier. The goals of this project are to test these hypotheses, define the mechanism(s) of Fhit-directed Chk1 termination and assess the in vivo and clinical relevance of these findings. To this end, we will examine the mechanism of Fhit modulation of Chk1 signaling using H1299 lung cancer and MKN74 stomach cancer cell lines. We hypothesize that Fhit promotes the ubiquitylation and degradation of pChk1. Next, we will confirm our mechanistic findings in a panel of cancer cell lines and determine the biological consequence of Fhit-deficiency and failed deactivation of Chk1 signaling in cancer cells. Lastly, we will examine a TMA of 300 Triple-Negative breast cancers for loss of Fhit expression and activation of the Chk1 pathway. This TMA will be linked to clinical features, such as disease-free survival, allowing for association of absent Fhit expression with clinical outcome. Results to be obtained will provide insight into the function of Fhit as a tumor suppressor, mechanisms of failed Chk1 deactivation and defects that contribute to cancer cell progression and survival of therapy. Importantly, the results will also help to define rationales for pre-clinical studies testing Chk1 and Parp1 inhibitors in combination with chemotherapy drugs for treatment of certain types of cancer, as well as criteria for identifying patients who will benefit from such therapies. PUBLIC HEALTH RELEVANCE: All cells have DNA repair systems and 'checkpoints' to make sure the cell does not survive if there has been too much damage, but cancer cells have over-activated 'checkpoints' that allow them to survive and grow even when they have not repaired all DNA damage. Loss of expression of the Fhit tumor suppressor protein enhances the ability of cancer cells to survive extensive DNA damage, even the damage caused by chemotherapy. This project will define the mechanism through which Fhit modulates the 'checkpoint', thus contributing to the understanding of aberrant 'checkpoints in cancer cells.