Determination of the role of CBP and p300 mediated Wnt signaling on colonic cells

描述(申请人提供)：Wnt信号通路，由激活的β-连环蛋白介导，负责启动大多数人类结直肠癌(CRC)病例，我们先前已经证明，组蛋白脱乙酰酶抑制剂(HDACis)，如丁酸盐，过度激活该通路可诱导CRC细胞死亡。介导Wnt信号激活效应的一个重要的细胞开关是β-连环蛋白与转录共激活因子CREB结合蛋白(CBP)和p300之间的关联的变化。CBP与β-连环蛋白的结合被认为激活了一系列与细胞增殖有关的基因，而p300介导的Wnt遗传程序被认为促进了细胞分化。已经发现小分子制剂通过改变CBP和P300与β-连环蛋白的结合来调节CBP/p300Wnt的转录程序。ICG-001和ICG-427分别抑制CBP和p300介导的Wnt活性，而IQ-1则阻止CBP介导的Wnt活性向p300介导的Wnt活性的转变。该方案的目的1旨在确定CBP和p300介导的Wnt信号在结直肠癌细胞对HDACis的反应中的作用。细胞将与HDACis和ICG-001、ICG-427或IQ-1共同处理，并测量Wnt活性、凋亡、增殖、分化和CBP-或p300-β-catenin结合水平。该建议的目的2是确定CBP/p300介导的Wnt活性在维持单个CRC细胞群体中高和低Wnt组分中的作用，这可能反映了在人类肿瘤中观察到的类似的异质性，并可能具有临床意义。目的3比较CBP和p300介导的Wnt活性在结直肠癌发生和发展中的作用，利用正常结肠癌细胞系CCD841CoN、腺瘤细胞系LT97、原发结肠癌细胞系SW480和淋巴结转移细胞系SW620。细胞将用HDACis和小分子试剂处理，并进行如上所述的检测。我们还将尝试利用CBP和p300介导的Wnt信号的变化来改变结肠癌细胞的细胞类型，改变CBP和p300介导的LT97腺瘤细胞系的基因表达，使腺瘤的表型转变为更具CCD-841CoN正常细胞或SW480癌细胞的特征。我们将利用微阵列分析来确定导致CBP或p300介导的结肠肿瘤表型变化的基因表达模式。这项研究产生的结果将导致未来更深入的项目，以进一步剖析CBP/p300Wnt介导的转录程序在结肠肿瘤中的作用，重点是调节这些遗传程序以实现化学预防效果的方法。 公共卫生相关性：Wnt信号通路负责启动大多数人类结直肠癌病例，我们之前已经证明，组蛋白脱乙酰酶抑制剂(HDACis)对该通路的过度激活可以诱导结直肠癌细胞死亡。由CBP或P300蛋白介导的WNT信号对癌细胞的生长、分化或死亡的决定有不同的影响，存在能特异性阻断CBP和P300介导的WNT信号的小分子药物。通过使用这些小分子抑制物，我们将确定CBP和p300如何影响结肠肿瘤的发生；这项研究的结果可以用来设计针对CBP和p300介导的Wnt活性的方法，以防止人类结直肠癌的发生和发展，降低与这种疾病相关的发病率和死亡率。

DESCRIPTION (provided by applicant): The Wnt signaling pathway, mediated through active beta-catenin, is responsible for initiating the majority of cases of human colorectal cancer (CRC), and we have previously shown that hyper-activation of this pathway by histone deacetylase inhibitors (HDACis), such as butyrate, can induce the death of CRC cells. An important cellular switch that mediates the effects of Wnt signaling activation is variation in the association between beta-catenin and the transcriptional coactivators CREB binding protein (CBP) and p300. Association of CBP with beta-catenin is thought to activate a set of genes linked to cell proliferation, while the p300-mediated Wnt genetic program is believed to promote cell differentiation. Small molecule agents have been discovered that modulate CBP/p300 Wnt transcriptional programs by altering the association of CBP and p300 to beta-catenin. ICG-001 and ICG-427 inhibit CBP and p300 mediated Wnt activity, respectively, while IQ-1 prevents the shift from CBP-mediated to a p300-mediated Wnt activity. Aim 1 of this proposal is designed to determine the role of CBP and p300 mediated Wnt signaling in the response of CRC cells to HDACis. Cells will be cotreated with HDACis and ICG-001, ICG-427, or IQ-1 and the levels of Wnt activity, apoptosis, proliferation, differentiation, and CBP- or p300-beta-catenin binding measured. Aim 2 of this proposal is to determine the role of CBP/p300-mediated Wnt activity in the maintenance of high- and low-Wnt fractions in single CRC cell populations, which may mirror similar heterogeneity observed in human tumors and which may be of clinical significance. Aim 3 will compare the effects of CBP and p300 mediated Wnt activity on CRC initiation and progression, utilizing CRC cell line model systems of initiation and progression: the normal colon cell lines CCD-841CoN, the adenoma line LT97, the primary colon carcinoma cell line SW480, and the lymph node metastasis cell line SW620. Cells will be treated with HDACis and the small molecule agents, and assayed as described above. We will also attempt to use changes in CBP and p300 mediated Wnt signaling to shift colonic cells between cell type, modifying CBP and p300 mediated gene expression in the LT97 adenoma line to shift the adenoma phenotype to more characteristic of the CCD- 841CoN normal cells, or the SW480 carcinoma cells. We will utilize microarray analyses to determine the patterns of gene expression responsible for these CBP or p300 mediated changes in colonic neoplastic phenotype. The findings generated from this study will lead to future, more in-depth projects to further dissect the action of CBP/p300 Wnt-mediated transcriptional programs in colonic neoplasia, with an emphasis on methods to modulate these genetic programs for chemopreventive effect. PUBLIC HEALTH RELEVANCE: The Wnt signaling pathway is responsible for initiating the majority of cases of human colorectal cancer, and we have previously shown that hyper-activation of this pathway by histone deacetylase inhibitors (HDACis) can induce the death of colorectal cancer cells. Wnt signaling mediated by the proteins CBP or p300 differentially influence decisions of cancer cell growth, differentiation, or death, and small molecule agents exist that can specifically block CBP and p300 mediated Wnt signaling. Through the use of these small molecule inhibitors, we will determine how CBP and p300 influence colonic tumorigenesis; the findings of this study can be utilized to devise methodologies, targeting CBP and p300 mediated Wnt activity, to prevent the development and progression of human colorectal cancer, decreasing the morbidity and mortality associated with this disease.