Development of novel miRNA based novel therapeutics for metastatic colorectal cancer

项目摘要/摘要本应用程序的目标是研究miR-15a在结直肠癌中的作用 并开发新型miR-15a模拟物作为治疗晚期转移性结直肠癌的潜在药物 男性和女性退伍军人都患有癌症。尽管在早期发现和改善治疗方面取得了进步 战略，美国仍有50,260人死于结直肠癌，其发病率为 在退伍军人中的比例也同样高。对氟嘧啶类药物的耐药性是主要原因之一。 晚期转移性结直肠癌治疗失败的原因。最近人们认识到， 表观遗传改变在肿瘤发生和5-氟尿嘧啶耐药中的关键作用 化疗。由于结直肠癌细胞高度异质性，化疗可能相当有效。 在消灭大多数快速增殖的癌细胞方面。然而，一小部分种群增长缓慢 癌症干细胞具有高度的抗药性，这会导致癌症复发。虽然它的作用机制 化疗耐药是复杂的，并且通常与靶标酶胸苷合成酶(TS， Tyms)，我们实验室最近的研究表明，表观遗传变化，如 非编码miRNAs的表达是这种5-FU耐药机制的主要贡献者，它通过提供 蛋白质合成在转录后和翻译水平上的急剧变化。MiRNAs是一类 具有重要调节功能的非编码小RNA。我们已经确定了一些与肿瘤有关的miRNAs 结直肠癌中的抑制功能。特别是，我们已经证明了miR-15a(hsa-miR-15a-5p) 作为一种有效的肿瘤抑制因子，可以通过抑制表达来克服结直肠癌的化疗耐药 几个主要的治疗靶基因(BMI1、BCL2、YAP1、DCLK1)及其相关通路。更多 重要的是，我们最近开发了一种新的策略来创建具有增强的修改的miRNA模拟物 在保留靶点特异性的同时消除5-FU耐药结肠癌干细胞的稳定性和有效性。 通过用5-氨基取代尿嘧啶(U)碱基来修饰miR-15a的靶链，设计了miR-15a模拟物。 赋在不同的地方。这种方法背后的原理是miR-15a的5-FU修饰将增强 稳定性，并将5-FU和多靶点miR-15a的力量结合到一个实体中，以创建治疗性 协同作用，因为miR-15a最终会分解为释放5-FU。5-FU修饰的miR的一个独特特征-- 15A是它可以在没有任何输送载体的情况下被结肠癌细胞内化。这代表着一个重大的 基于miRNA的治疗开发的进展和范式转变。我们的初步结果显示 这种修饰提高了miR-15a模拟物的效力和稳定性，并增强了其 体内抑制结肠癌转移，未观察到任何毒性。具体目标1：我们将定义直接 在结肠癌中模拟miR-15a和miR-15a的靶点和途径并研究miR-15a的作用 在结肠癌细胞和3D有机物中模拟细胞凋亡、细胞周期控制和化疗耐药。特定的 目的：研究miR-15a模拟物在体内的毒性、药代动力学和组织分布。 癌症小鼠模型。具体目标3：我们将开发和表征miR-15a的治疗潜力 用体内结肠癌小鼠模型模拟转移性结直肠癌。

Project Summary/Abstract The goal of this application is to investigate the roles of miR-15a in colorectal cancer and to develop novel miR-15a mimic as potential therapeutics to treat advanced metastatic colorectal cancer in both male and female Veterans. Despite advancements in early detection and improved treatment strategies, there are still 50,260 deaths due to colorectal cancer in the United States, and its incidence is equally high in the Veteran population. Resistance to fluoropyrimidine-based chemotherapy is one of the major causes for the failure of treating advanced metastatic colorectal cancer. It has been recognized recently that epigenetic alterations play a key role in tumorigenesis and resistance to 5-fluorouracil (5-FU) based chemotherapy. Because colorectal cancer cells are highly heterogeneous, chemotherapy can be quite effective in eliminating most of the rapid proliferating cancer cells. However, a small population of slow proliferating cancer stem cells are highly resistant which leads to cancer recurrence. Although the mechanism of chemoresistance is complex and is often associated with elevated target enzyme thymidylate synthase (TS, TYMS), recent studies from our laboratory have shown that epigenetic alterations such as changes in expression of non-coding miRNAs are major contributors to such resistance mechanisms to 5-FU by providing acute changes in protein synthesis at the post-transcriptional and translational levels. miRNAs are a class of small non-coding RNAs with crucial regulatory functions. We have identified a number of miRNAs with tumor suppressive functions in colorectal cancer. In particular, we have demonstrated that miR-15a (hsa-miR-15a-5p) can overcome chemoresistance in colorectal cancer as a potent tumor suppressor by inhibiting the expression of several major therapeutic target genes (BMI1, BCL2, YAP1, DCLK1) and associated pathways. More importantly, we have recently developed a novel strategy to create modified miRNA mimics with enhanced stability and efficacy for eliminating 5-FU resistant colon cancer stem cells while retaining target specificity. miR-15a mimics were designed by modifying the target strand of miR-15a by replacing uracil (U) bases with 5- FU at various locations. The rationale behind this approach is that 5-FU modification of miR-15a will enhance stability, and also combining the power of 5-FU and multi-targeted miR-15a into one entity to create therapeutic synergy, as miR-15a will breakdown eventually to release 5-FU. A unique feature of the 5-FU modified miR- 15a is that it can be internalized by colon cancer cells without any delivery vehicle. This represents a major advancement and a paradigm shift in miRNA based therapeutic development. Our preliminary results show that such modification improves the potency and stability of the miR-15a mimic and enhances its ability to inhibit colon cancer metastasis in vivo without any observed toxicity. Specific Aim 1: We will define the direct targets and pathways of miR-15a and miR-15a mimic in colon cancer and characterize the effects of miR-15a mimic on apoptosis, cell cycle control, and chemoresistance in colon cancer cells and 3D organoids. Specific Aim 2: We will investigate the toxicity, pharmacokinetics and tissue distribution of miR-15a mimic in vivo colon cancer mouse models. Specific Aim 3: We will develop and characterize the therapeutic potential of miR-15a mimic in metastatic colorectal cancer using in vivo colon cancer mouse models.