The role of ATOH1 as a tumor suppressor in colorectal cancer

描述(申请人提供)：Notch-Atoh1分子通路在结直肠癌(CRC)肿瘤发生中的重要性最近被认识到，表明该通路是新的结直肠癌治疗的靶点。我们的初步数据表明，Atoh1是Notch定向的肠道干细胞分化计划的关键守门人，在结直肠癌中，Atoh1发挥着肿瘤抑制因子的作用，在结直肠癌中，它被甲基化和缺失沉默。此外，我们的数据显示，Notch抑制药物--迫使结肠细胞分化和阻止增殖的药物--需要Atoh1来发挥这些作用。我们还确定SPDEF是Atoh1的下游靶点，并表明SPDEF抑制肠上皮细胞的增殖。综上所述，这些结果提示Atoh1可能是Notch途径通过SPDEF调控癌细胞分化和增殖的关键靶点。这项工作将检验这一假设，即Atoh1是一种结肠癌抑制因子，通过SPDEF介导Notch抑制剂的抗癌作用。在目标1中，我们将确定Atoh1沉默在肿瘤启动中的作用及其与突变型CRC表型的关系。我们将确定大肠癌中Atoh1沉默是否需要突变或甲基化，并确定由我们在范德比尔特大学的合作者提供的原代人类肿瘤样本中Atoh1沉默的CRC的分子表型。我们将通过量化Atoh1突变的结肠上皮细胞在致癌剂治疗后的早期肿瘤变化，直接测试Atoh1突变是否有助于转基因小鼠的肿瘤启动。在目标2中，我们将测试Notch抑制剂(GSI)在结肠癌中的抗癌作用是否需要Atoh1。GSIS代表了一种令人兴奋但机械上定义较少的癌症治疗方法。我们预测，Atoh1沉默将阻止GSI治疗在大肠癌中发挥作用。我们将在小鼠肿瘤和具有多种癌症表型的癌细胞系中测试GSI的抗增殖作用是否需要Atoh1。我们还将测试一种新的治疗方法，通过重新表达Atoh1来恢复Atoh1沉默的细胞对GSI的敏感性。在目标3中，我们将通过验证Atoh1通过其靶转录因子SPDEF引导细胞周期退出的假说来确定Atoh1的S抑瘤作用的机制。在这里，我们将在结肠癌细胞系和转基因小鼠中使用互补的方法来确定SPDEF是否指导细胞周期停滞。同样，我们将定义SPDEF诱导介导Atoh1/GSI诱导的基因敲除小鼠和CRC细胞生长停滞的要求。癌是一个主要的公共卫生负担，需要在了解癌症分子发病机制的基础上进行新的治疗。该项目的结果将：1)确定一种新的亚型-Atoh1沉默的CRC-可能对GSI治疗无效；2)确定Atoh1沉默的机制和恢复Atoh1沉默的CRC对GSI的敏感性的策略；3)确定Atoh1肿瘤抑制和下游治疗靶点(SPDEF)的机制。因此，我们将确定一种新的抗癌治疗方法--GSI通过Atoh1：SPDEF诱导生长抑制--的分子机制。 公共卫生相关性：该项目与美国国立卫生研究院减轻疾病和残疾负担的使命相关，作为研究人类疾病的原因、预防和治疗以及人类成长和发展过程的组成部分。拟议的研究对公众健康很重要，因为它们将影响结直肠癌的诊断和治疗，并深入了解肠道内稳态的正常途径。

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.