The role of ATOH1 as a tumor suppressor in colorectal cancer

描述（由申请人提供）：Notch-Atoh 1分子途径在结直肠癌（CRC）肿瘤发生中的重要性最近得到了认可，表明该途径是新CRC治疗的靶点。我们的初步数据表明，ATOH 1是Notch指导的肠干细胞分化程序的关键守门人，并且ATOH 1在CRC中作为肿瘤抑制因子发挥作用，其中它通过甲基化和缺失而沉默。此外，我们的数据表明，Notch抑制药物-这迫使分化和阻断结肠细胞增殖-需要ATOH 1的这些影响。我们还确定了SPDEF作为ATOH 1的下游靶点，并表明SPDEF抑制肠上皮细胞增殖。综上所述，这些结果表明ATOH 1可能是Notch途径通过SPDEF调节CRCs分化和增殖的关键靶标。这项工作将测试ATOH 1是结肠肿瘤抑制因子的假设，其通过SPDEF介导Notch抑制剂的抗癌作用。在目标1中，我们将确定ATOH 1沉默在肿瘤发生中的作用及其与突变CRC表型的相关性。我们将确定CRC中ATOH 1沉默是否需要突变或甲基化，并确定由我们的合作者在范德比尔特大学提供的原发性人类肿瘤样本中ATOH 1沉默CRC的分子表型。我们将通过量化致癌物治疗后Atoh 1突变结肠上皮细胞的早期肿瘤变化，直接测试Atoh 1突变是否有助于转基因小鼠的肿瘤发生。在目标2中，我们将测试ATOH 1是否是Notch抑制剂（GSI）在结肠癌中的抗癌作用所必需的。GSI代表了一种令人兴奋但机制上定义较少的治疗癌症的方法。我们预测ATOH 1沉默将阻止GSI治疗在CRC中起作用。我们将测试Atoh 1是否是GSI在小鼠肿瘤和具有多种确定的癌症表型的癌细胞系中的抗增殖作用所必需的。我们还将测试一种新型治疗方法，通过重新表达ATOH 1来恢复ATOH 1沉默细胞对GSI的敏感性。在目标3中，我们将通过测试ATOH 1通过其靶转录因子SPDEF指导细胞周期退出的假设来定义ATOH 1肿瘤抑制功能的机制。在这里，我们将在结肠癌细胞系和转基因小鼠中使用互补方法来确定SPDEF是否指导细胞周期停滞。同样，我们将定义SPDEF诱导介导敲除小鼠和CRC细胞中ATOH 1/GSI诱导的生长停滞的要求。CRC构成了主要的公共卫生负担，需要基于对癌症分子发病机制的理解的新疗法。本项目的结果将：1）鉴定一种新的亚型-ATOH 1沉默的CRC-其可能对GSI治疗难治; 2）确定ATOH 1沉默的机制和在ATOH 1沉默的CRC中恢复GSI敏感性的策略; 3）鉴定ATOH 1肿瘤抑制的机制和下游治疗靶点（SPDEF）。因此，我们将确定一种新的方法-GSI通过ATOH 1：SPDEF诱导生长停滞的抗癌治疗的分子机制。 公共卫生相关性：该项目与NIH的使命有关，即减少疾病和残疾的负担，作为研究人类疾病的原因、预防和治疗以及人类生长和发展过程的一个组成部分。拟议的研究对公共卫生很重要，因为它们将影响结直肠癌的诊断和治疗，并深入了解肠道稳态的正常途径。

DESCRIPTION (provided by applicant): The importance of the Notch-Atoh1 molecular pathway in colorectal cancer (CRC) tumorigenesis has recently being recognized, suggesting that this pathway is a target for new CRC therapeutics. Our preliminary data suggest that ATOH1 is a critical gatekeeper for the program of Notch-directed differentiation of intestinal stem cells, and that ATOH1 functions as a tumor suppressor in CRC where it is silenced by methylation and deletion. Furthermore, our data show that Notch inhibitory drugs-which force differentiation and block proliferation in colon cells-require ATOH1 for these effects. We also identified SPDEF as a downstream target of ATOH1 and showed that SPDEF inhibits intestinal epithelial proliferation. Taken together, these results suggest that ATOH1 may be the key target of the Notch pathway regulating differentiation and proliferation in CRCs via SPDEF. This work will test the hypothesis that ATOH1 is a colon tumor suppressor that mediates anti- cancer effects of Notch inhibitors via SPDEF. In Aim 1, we will determine the role for ATOH1 silencing in tumor initiation and its association with mutator CRC phenotypes. We will determine whether ATOH1 silencing in CRC requires mutation or methylation and define the molecular phenotype of ATOH1-silenced CRCs in primary human tumor samples provided by our collaborators at Vanderbilt University. We will directly test if Atoh1 mutation contributes to tumor initiation in transgenic mice, by quantifying early neoplastic changes in Atoh1- mutant colonic epithelia following carcinogen treatment. In Aim 2, we will test whether ATOH1 is required for the anti-cancer effect of Notch inhibitors (GSIs) in colon cancer. GSIs represent an exciting but mechanistically less defined approach to treat cancer. We predict that ATOH1 silencing will prevent GSI therapy from working in CRCs. We will test whether Atoh1 is required for the anti-proliferative effect of GSIs, both in mouse tumors and in cancer cell lines with multiple defined cancer phenotypes. We will also test a novel therapeutic approach to restore sensitivity to GSIs in ATOH1-silenced cells by re-expression of ATOH1. In Aim 3, we will define the mechanism of ATOH1's tumor suppressive function by testing the hypothesis that ATOH1 directs cell cycle exit via its target transcription factor SPDEF. Here, we will use complementary approaches in colon cancer cell lines and transgenic mice to determine whether SPDEF directs cell cycle arrest. Similarly, we will define the requirement for SPDEF induction to mediate ATOH1/GSI-induced growth arrest in knockout mice and CRC cells. CRCs constitute a major public health burden, with a need for novel therapies based on understanding of the molecular pathogenesis of cancer. The results of this project will: 1) identify a novel subtype-ATOH1- silenced CRC-which are likely refractory to GSI treatment; 2) define the mechanism of ATOH1 silencing and strategies to restore GSI sensitivity in ATOH1-silenced CRCs; 3) identify the mechanism of ATOH1 tumor suppression and downstream therapeutic targets (SPDEF). Thus, we will define the molecular mechanisms underpinning a new approach-GSI-induced growth arrest via ATOH1:SPDEF-to anti-cancer therapy. PUBLIC HEALTH RELEVANCE: This project has relevance for the NIH's mission to reduce the burdens of illness and disability, as a component of research into the causes, prevention, and cure of human diseases, and the processes of human growth and development. The proposed studies are important to public health because they will impact the diagnosis and treatment of colorectal cancers, and give insight into the normal pathway of intestinal homeostasis.