Development of novel miRNA based novel therapeutics for metastatic colorectal cancer

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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.