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，因此肿瘤前病变显示出激活的DDR检查点，其阻碍细胞增殖并诱导细胞衰老和/或凋亡。巧合的是，DNA损伤检查点已被认为对癌细胞存活是重要的，这是由于这些细胞所引起的DNA损伤的程度，无论是内源性的还是通过DNA靶向治疗，以及需要修复以从损伤中恢复。最近有人提出，Chk 1激酶（S/G2检查点信号）的降解机制缺陷可能导致癌细胞对某些抗癌药物的敏感性丧失。有趣的是，FHIT，一种复制应激靶点，在几种类型的癌症中经常发生改变，这种改变导致Fhit蛋白表达的丧失。此外，已显示癌细胞中Fhit的再表达调节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缺陷癌细胞表现出异常的DNA损伤检查点反应和延长的S期Chk 1激活，而暴露于DNA复制应激。这些观察结果意味着一个以前未知的作用Fhit作为参与者在持续复制叉块和/或广泛的DNA损伤的细胞中的信号Chk 1失活。基于这些发现，我们假设Fhit定向的Chk 1终止使癌细胞对DNA靶向抗癌药物敏感，并且肿瘤中Fhit表达的丧失是对遗传毒性剂无反应且对DNA损伤不敏感的细胞的特征。Fhit的这种肿瘤抑制功能用于消除具有累积DNA损伤的癌细胞，因此可能有助于DDR肿瘤发生屏障。本项目的目标是测试这些假设，定义Fhit介导的Chk 1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MKN 74胃癌细胞系来研究Fhit调节Chk 1信号传导的机制。我们推测Fhit促进了pChk 1的泛素化和降解。接下来，我们将在一组癌细胞系中证实我们的机制发现，并确定癌细胞中Fhit缺陷和Chk 1信号转导失活失败的生物学后果。最后，我们将检查300例三阴性乳腺癌的TMA，以确定Fhit表达的缺失和Chk 1通路的激活。该TMA将与临床特征（例如无病生存期）相关联，从而允许Fhit表达缺失与临床结果相关联。获得的结果将提供深入了解Fhit作为肿瘤抑制因子的功能，Chk 1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临床前研究的基本原理，这些研究测试Chk 1和Parp 1抑制剂与化疗药物联合治疗某些类型的癌症，以及确定从这些治疗中受益的患者的标准。 公共卫生相关性：所有细胞都有DNA修复系统和“检查点”，以确保细胞在受到太多损伤时无法存活，但癌细胞已经过度激活了“检查点”，即使它们没有修复所有DNA损伤，也可以存活和生长。Fhit肿瘤抑制蛋白表达的缺失增强了癌细胞在广泛的DNA损伤中存活的能力，甚至是化疗引起的损伤。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.