MicroRNAs (miRNAs or miRs) have a critical role in regulating stem cells (SCs) during development and altered expression can cause developmental defects and/or disease. Indeed, aberrant miRNA expression leads to wide-spread transcriptional dysregulation which has been linked to many cancers. Mounting evidence also indicates a role for miRNAs in the development of the cancer SC (CSC) phenotype. Our goal herein is to provide a review of: (i) current research on miRNAs and their targets in colorectal cancer (CRC), and (ii) miRNAs that are differentially expressed in colon CSCs. MicroRNAs can work in clusters or alone when targeting different SC genes to influence CSC phenotype. Accordingly, we discuss the specific miRNA cluster classifications and isomiRs that are predicted to target the ALDH1, CD166, BMI1, LRIG1, and LGR5 SC genes. miR-23b and miR-92A are of particular interest because our previously reported studies on miRNA expression in isolated normal versus malignant human colonic SCs showed that miR-23b and miR-92a are regulators of the LGR5 and LRIG1 SC genes, respectively. We also identify additional miRNAs whose expression inversely correlated with mRNA levels of their target genes and associated with CRC patient survival. Altogether, our deliberation on miRNAs, their clusters, and isomiRs in regulation of SC genes could provide insight into how dysregulation of miRNAs leads to the emergence of different CSC populations and SC overpopulation in CRC.
One reason for lack of efficacy in cancer therapeutics is tumor heterogeneity. We hypothesize that tumor heterogeneity arises due to emergence of multiple cancer stem cell (CSC) subpopulations because miRNAs regulate expression of stem cell genes in CSCs. Our goal was to determine if: i) multiple CSC subpopulations exist in a human CRC cell population, and ii) miRNAs are differentially expressed in the different CSC subpopulations. We discovered that at least four different CSC populations (ALDH1, CD166, LGR5, LRIG1) exist in the HT29 cell line. CSC subpopulations were quantified using co-staining for multiple stem cell markers, isolated using FACS, and analyzed by NanoString miRNA profiling. The miRNA expression pattern in each CSC subpopulation was analyzed relative to miRNA expression patterns in other CSC subpopulations. Messenger RNAs predicted to be targeted by the upregulated miRNAs in each CSC subpopulation were: 1) identified using bioinformatics analyses, and 2) classified according to their predicted functions using David functional annotation analyses. We found multiple CSC subpopulations with a unique miRNA signature in each CSC subpopulation. Notably, the miRNAs expressed within one CSC subpopulation are predicted to target and downregulate the CSC genes and pathways that establish the other CSC subpopulations. Moreover, mRNAs predicted to be targeted by miRNAs in the different CSC subpopulations have different cellular functional classifications. That different CSC subpopulations express miRNAs that are predicted to target CSC genes expressed in other CSC subpopulations provides a mechanism that might explain the co-existence of multiple CSC subpopulations, tumor heterogeneity, and cancer therapy resistance.
Our research goal is to discover the molecular mechanisms behind tumor heterogeneity in CRC. We previously showed that CRCs contain multiple subpopulations of cancer stem cells (CSCs) which may explain the occurrence tumor heterogeneity and resistance to treatment. To determine how CSC sub-populations might arise, we are studying miRNA expression in CRC SCs. MicroRNAs are known to regulate SC phenotype and are found to be dysregulated in many cancers. Hypothesis: Tumor heterogeneity results from existence of multiple CSC subpopulations that are regulated by distinct miRNAs. Accordingly, we are using bioinformatics and miRNA profiling to identify miRNAs that target SC genes in CRCs. Indeed, our miRNA expression profiling of normal and malignant ALDH+ human colonic SCs showed that miRNA92a targets the SC gene LRIG1 and upregulation of miRNA92a leads to decreased LRIG1 expression. We also discovered that miRNA23b targets the SC gene LGR5 and miRNA23b is upregulated in ALDH+ CSCs. We have identified several other candidate miRNAs that are predicted to target CD166, ALDH1A1, BMI1, LRG5, and LRIG1 SC genes. We are currently in the process of validating whether these miRNAs contribute to emergence of specific CSC sub-populations in CRCs. Thus, identifying miRNAs that regulate CSC subpopulations should provide new strategies to modulate CSC composition in order to sensitize tumors to treatments. Citation Format: Victoria A. Stark, Vignesh Viswanathan, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Identifying differentially expressed miRNAs in CRC stem cell subpopulations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2360.
We investigated miRNAs that are implicated to target genes that encode different stem cell markers (SC) during tumorigenesis of colorectal cancer (CRC), one of the leading causes of cancer deaths in the US. One reason behind the lack of efficacy in cancer drug therapeutics is due to the development of tumor heterogeneity. We hypothesize that different miRNAs target different SC genes, which leads to the emergence of multiple cancer SC (CSC) subpopulations and tumor heterogeneity. Flow cytometry helped evaluate the expression of multiple SC markers including ALDH1, CD166, LGR5, and LRIG1 in the HT29 CRC cell line. CSC sub-populations were isolated using fluorescence activated cell sorting (FACS) and the expression of miRNAs in each CSC subpopulation was measured using NanoString profiling. The differential expression of miRNAs between CSC subpopulations helped rank the different levels of miRNA upregulation in each of the CSC subpopulations. The mRNAs predicted to be targeted by the most significantly upregulated miRNAs were identified using bioinformatics analysis via NanoString and mRNAs were classified according to their predicted function by David functional annotation analysis. Altogether, our results indicate that multiple CSC subpopulations exist in the HT29 cell-line, and we identified unique miRNA signatures for each CSC subpopulation. Moreover, the mRNAs predicted to be targeted by the miRNAs in each CSC subpopulation were found to have different functional classifications. We conclude that multiple CSC subpopulations emerge in HT29 cells due to the expression of unique miRNAs that target different SC genes (and their co-expressed genes), resulting in the emergence of multiple CSC subpopulations and tumor heterogeneity. Citation Format: Victoria A. Stark, Caroline O. B. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the role of microRNAs that target stem cell genes in the emergence of tumor heterogeneity in development of human colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5823.
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