Abstract 3805: Revealing the role of microRNAs in the emergence of cancer stem cell heterogeneity during colorectal cancer progression

Abstract

Abstract Our goal is to identify microRNAs (miRNAs) that target mRNAs involved in stem cell (SC) signaling pathways involved in colorectal cancer (CRC) tumorigenesis. SC overpopulation drives CRC progression but exact mechanism is unclear. Hypothesis: specific miRNAs target SC genes, which leads to the emergence of multiple cancer SC (CSC) sub-populations and tumor heterogeneity. We used flow cytometry to identify CSC sub-populations based on expression of SC markers including ALDH1, LGR5 and others in the HT-29 CRC cell line. CSC sub-populations were isolated and measured using FACS and expression of miRNAs in each CSC sub-population was quantified using NanoString profiling. The level of miRNA upregulation in each CSC sub-population was ranked based on differential expression of the miRNAs between different CSC sub-populations. We chose to further analyze the LGR5 and ALDH SC sub-populations because they showed: i) the largest difference between CSC sub-population sizes, ii) the smallest size (<1%) of the co-positive (LGR5+/ALDH+) cell sub-population, iii) the highest numbers of differentially expressed miRNAs. Note that WNT signaling mainly occurs via LGR5+ CSC and retinoic acid (RA) signaling mainly occurs via ALDH+ CSCs. We report herein miRNAs that are differentially expressed between ALDH+/LGR5- compared to ALDH-/LGR5- subpopulation and LGR5+/ALDH- versus ALDH-/LGR5-. The mRNAs expressed in CSC subpopulations identified from Nanostring profiling were analyzed using bioinformatics for their predicted binding by upregulated miRNAs. Our findings show that: 1) multiple CSC subpopulations exist in the HT-29 cell population; 2) each CSC subpopulation has a unique miRNA signature. Notably, RXRA mRNA is significantly upregulated in the ALDH+/LGR5- CSC sub-population but not in the LGR5+/ALDH- CSC subpopulation. We identified 5 miRs upregulated in the LGR5+/ALDH- CSC subpopulation that are predicted to target the 3’UTR of RXRA mRNA. One of these miRs, miR-660-3p, is specifically upregulated in the LGR5+/ALDH- CSC subpopulation and binding of miR-660-3p to RXRA mRNA was validated using luciferase assay in HT-29 cells. We previously reported that RXRA is selectively expressed in ALDH + CSC sub-populations. Also, RXRA is known to bind with β-catenin leading to its degradation and suppression of WNT-based transcriptional activity. Taken together, these results indicate that expression of miR-660-3p, by inhibiting RXRA expression, increases WNT signaling and decreases RA signaling in LGR5+/ALDH- CSCs. Thus, upregulated miR-660-3p targets a key gene in the RA signaling pathway (RXRA) which could contribute to the emergence of SC sub-populations during CRC development. Citation Format: Molly Lausten, Victoria Stark, Caroline Facey, Lynn Opdenaker, Bruce M. Boman. Revealing the role of microRNAs in the emergence of cancer stem cell heterogeneity during colorectal cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3805.