MicroRNAs as Targets for Colon Cancer Chemotherapy

描述(申请人提供)：结直肠癌是包括美国在内的大多数发达国家的主要死亡原因之一。结肠癌化疗依赖于许多细胞毒药物、靶向药物及其组合，因此越来越需要开发针对特定途径的替代药物，以抑制肿瘤的生长、进展和转移并诱导细胞凋亡。特异性蛋白(SPS)是一类在许多肿瘤中过表达的转录因子，调节肿瘤细胞和肿瘤生长(p27抑制)、生存(Survivin)和血管生成(VEGFR1和VEGFR2)所需基因的表达。本实验室的研究表明，托芬那酸(TA)和白桦酸(BA)以及一种新的人工合成的三萜酸(酯)methyl-2-cyano-3，11-dioxo-18¿-olean-1，12-dien-30-oate(CDoda-Me)可诱导结肠癌细胞G2/M期生长停滞和Sp蛋白的非蛋白酶体降解。这些效应直接与化合物诱导的致癌microRNA-27A(mir-27A)和其他miRs的表达调控有关。因此，我们假设TA、BA和CDoda-Me代表了一类独特的针对miR-27A和其他miR的抗癌药物。这项拟议的研究将描述药物-miR相互作用在结肠癌中的作用机制和效果。目的1重点研究TA-/BA-/CDoda-Me-miR-27A的相互作用，研究依赖miR-27A的ZBTB10和MYT-1表达的激活及其对Sp和Sp依赖基因的下游调控，以及对结肠癌细胞的生长抑制、抗血管生成和促凋亡反应。TA、BA和CDoda-Me还降低了结肠癌细胞中其他miR的表达，其中包括与miR-27a形成簇的miR-23a和miR-24-2。目的2研究TA-、BA-和CDODA-Me-miR(23a~24-2)之间的相互作用，并确定它们在这些化合物的抗癌活性中的作用。目的研究TA、BA和CDodA-Me在小鼠移植瘤和Min结肠癌模型中的体内抗癌活性，并确定化合物与miR的相互作用。此外，已经发展了过表达miR-27A的小鼠作为探针，以研究这种致癌miR在结肠癌发生中的作用。这些研究将为TA、BA和CDoda-Me作为一类新的抗癌药物的疗效和作用机制提供关键数据，这些药物通过多种途径发挥作用，包括直接作用于microRNAs及其相关基因转录本。与公共健康相关：本实验室最近的研究已经确定了两种结构类型的化合物，它们在癌症中诱导Sp蛋白抑制，并抑制癌细胞的生长和存活。这些化合物包括三萜类化合物白桦酸(BA)和甲基2-cyano-3，11-dioxo-18¿-olean-1，12-dien-30-oate(CDoda-Me)和非甾体抗炎药托芬那酸(TA)。本项目中提出的研究将集中在这些抗癌药物的作用机制上，重点是它们对致癌microRNA-27A(miR-27A)和其他miR的影响，以及在Sp蛋白和其他关键基因的抑制中的作用。

DESCRIPTION (provided by applicant): Colorectal cancer is one of the leading causes of death in most developed countries including the United States. Colon cancer chemotherapy relies on a number of cytotoxic drugs, targeted agents and their combinations, and there is an increasing need to develop alternative drugs targeting specific pathways that inhibit tumor growth, progression and metastasis and induce apoptosis. Specificity proteins (Sps) are transcription factors overexpressed in many tumors, and Sps regulate expression of genes required for cancer cell and tumor growth (p27 suppression), survival (survivin), and angiogenesis (VEGF, VEGFR1 and VEGFR2). Studies in this laboratory have now shown that tolfenamic acid (TA) and betulinic acid (BA) and a novel synthetic triterpenoid acid (ester), namely methyl-2-cyano-3,11-dioxo-18¿-olean-1,12-dien-30-oate (CDODA-Me) induce G2/M growth arrest and proteasome-independent degradation of Sp proteins in colon cancer cells. These effects are directly linked to compound-induced modulation of expression of oncogenic microRNA-27a (mir-27a) and other miRs. Therefore, we hypothesize that TA, BA and CDODA-Me represent a unique class of anticancer agents that target miR-27a and other miRs. The proposed studies will characterize the mechanisms of action and effects resulting from drug-miR interactions in colon cancer. Aim 1 will focus on TA-/BA-/CDODA-Me-miR-27a interactions and investigate the activation of miR-27a-dependent ZBTB10 and Myt-1 expression and their subsequent downstream modulation of Sp and Sp-dependent genes, growth inhibitory, antiangiogenic and proapoptotic responses in colon cancer cells. TA, BA and CDODA-Me also decrease expression of other miRs in colon cancer cells, and these include miR-23a and miR-24-2 which form a cluster with miR-27a. Aim 2 will investigate TA-, BA- and CDODA-Me-miR(23a~24-2) interactions and determine their role in mediating the anticarcinogenic activities of these compounds. Aim 3 will investigate the in vivo anticarcinogenic activity of TA, BA and CDODA-Me in a mouse xenograft and "Min" model for colon cancer and determine the compound-miR interactions. In addition, mice overexpressing miR-27a have been developed as probes for investigating the role of this oncogenic miR in colon carcinogenesis. These studies will provide critical data on the efficacy and mechanisms of action of TA, BA and CDODA-Me as a novel class of anticancer drugs that act through multiple pathways including direct effects on microRNAs and their associated gene transcripts. PUBLIC HEALTH RELEVANCE: Recent studies in this laboratory have identified two structural classes of compounds that induce Sp protein repression in cancers and inhibit cancer cell growth and survival. These compounds include the triterpenoids betulinic acid (BA) and methyl 2-cyano-3,11-dioxo-18¿-olean-1,12-dien-30-oate (CDODA-Me) and the NSAID tolfenamic acid (TA). The studies proposed in this project will focus on the mechanisms of action of these anticancer agents with emphasis on their effects on the oncogenic microRNA-27a (miR-27a) and other miRs and the role in repression of Sp proteins and other critical genes.