Development of novel miRNA based novel therapeutics for metastatic colorectal cancer

本申请的目的是研究miR-15 a在结直肠癌中的作用。 癌症和开发新的miR-15 a模拟物作为治疗晚期转移性结直肠癌的潜在疗法 男性和女性退伍军人的癌症。尽管在早期发现和改善治疗方面取得了进展 尽管采取了这些策略，但在美国仍有50，260人死于结直肠癌，其发病率为 在退伍军人中也同样高。对氟尿嘧啶为基础的化疗耐药是主要的 晚期转移性结直肠癌治疗失败的原因。最近人们认识到， 表观遗传学改变在肿瘤发生和对5-氟尿嘧啶（5-FU）耐药中起关键作用， 化疗因为结直肠癌细胞是高度异质性的，所以化疗可能相当有效。 消灭大部分快速增殖的癌细胞。然而，一小部分缓慢繁殖的 癌症干细胞具有高度抗性，这导致癌症复发。虽然， 化学抗性是复杂的并且通常与升高的靶酶胸苷酸合酶（TS， TYMS），我们实验室最近的研究表明，表观遗传改变，如 非编码miRNA的表达是5-FU耐药机制的主要贡献者， 转录后和翻译水平的蛋白质合成的急剧变化。miRNAs是一类 具有重要调控功能的小的非编码RNA。我们已经鉴定了许多与肿瘤相关的miRNAs， 结肠直肠癌的抑制功能。特别是，我们已经证明miR-15 a（hsa-miR-15 a-5 p） 作为一种有效的肿瘤抑制因子，通过抑制 几个主要的治疗靶基因（BMI 1，BCL 2，YAP 1，DCLK 1）和相关途径。更 重要的是，我们最近开发了一种新的策略来创建修饰的miRNA模拟物， 在保持靶特异性的同时消除5-FU抗性结肠癌干细胞的稳定性和功效。 miR-15 a模拟物是通过用5-氨基嘧啶（U）碱基替换尿嘧啶（U）碱基来修饰miR-15 a的靶链而设计的。 在不同的地点。这种方法背后的基本原理是miR-15 a的5-FU修饰将增强miR-15 a的表达。 稳定性，并且还将5-FU和多靶向miR-15 a的能力组合到一个实体中以产生治疗效果。 这是因为miR-15 a最终将分解释放5-FU。5-FU修饰的miR-21的一个独特特征是， 15 a的另一个优点是它可以在没有任何递送载体的情况下被结肠癌细胞内化。这代表了一个重大的 这是基于miRNA的治疗开发的进步和范式转变。我们的初步结果表明 这种修饰改善了miR-15 a模拟物的效力和稳定性，并增强了其 在体内抑制结肠癌转移而没有任何观察到的毒性。具体目标1：我们将定义直接 miR-15 a和miR-15 a模拟物在结肠癌中的靶点和途径，并表征miR-15 a的作用 在结肠癌细胞和3D类器官中模拟细胞凋亡、细胞周期控制和化学抗性。具体 目的2：研究miR-15 a模拟物在结肠的毒性、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-15 a的治疗潜力 在转移性结肠直肠癌中使用体内结肠癌小鼠模型进行模拟。

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.