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）被认为是肿瘤发生的屏障，因此肿瘤前病变显示激活的DDR检查点，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存活很重要，因为这些细胞所遭受的DNA损伤的程度，无论是内源性的还是DNA靶向治疗的，以及从损伤中恢复的修复需要。最近有人提出，信号信号S/G2检查点的Chk1激酶降解机制的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是一种复制应激靶点，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，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，当暴露于DNA复制应激时，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Chk1激活。这些观察结果表明，在持续复制叉阻断和/或广泛DNA损伤的细胞中，Fhit作为Chk1失活信号的参与者，其作用是以前未知的。基于这些发现，我们假设Fhit导向的Chk1终止使癌细胞对靶向DNA的抗癌药物敏感，并且肿瘤中Fhit表达的缺失是对基因毒性药物无反应且对DNA损伤不敏感的细胞的特征。Fhit的抑瘤功能有助于消除累积DNA损伤的癌细胞，因此可能有助于DDR肿瘤发生屏障。该项目的目标是验证这些假设，确定fhit介导的Chk1终止的机制，并评估这些发现的体内和临床相关性。为此，我们将利用H1299肺癌和MKN74胃癌细胞系研究Fhit调控Chk1信号通路的机制。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证实我们的机制发现，并确定fhit缺乏和癌细胞中Chk1信号失活失败的生物学后果。最后，我们将在300例三阴性乳腺癌中检测Fhit表达缺失和Chk1通路激活的TMA。这种TMA将与临床特征相关联，如无病生存，允许Fhit表达缺失与临床结果相关联。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，该结果还将有助于确定临床前研究的基本原理，测试Chk1和Parp1抑制剂与化疗药物联合治疗某些类型的癌症，以及确定将从此类治疗中受益的患者的标准。

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.