Abstract
Cancer stem cells (CSCs) play a crucial role in colorectal cancer by sustaining intratumoral heterogeneity, therapeutic resistance, and metastatic potential. CD133 (PROM1) is among the most frequently used surface markers for CSC identification, whereas TRIM28, a versatile epigenetic regulator, has been implicated in controlling CD133 expression and stem-like features. In this study, we performed a detailed molecular and functional analysis of Caco2 colorectal cancer cell clones with individual knockouts of CD133 or TRIM28. Elimination of CD133 neither altered global gene expression, as confirmed by transcriptome profiling, nor affected key cellular properties. In contrast, loss of TRIM28 led to a marked reduction in CD133 protein abundance and induced extensive molecular and phenotypic remodeling. TRIM28 knockout was associated with broad transcriptomic changes involving more than 500 differentially expressed genes, decreased proliferative activity monitored by time-lapse imaging, and reduced sensitivity to paclitaxel, cisplatin, and curcumin. Furthermore, immune evasion molecules CD24 and CD47 ("don't eat me" signals) were strongly upregulated in TRIM28-deficient cells, consistently confirmed by both RNA-Seq and flow cytometry analyses. At the same time, imaging flow cytometry and mitochondrial activity assays indicated that these effects were not due to major shifts in mitotic index or bioenergetic status. Altogether, our results demonstrate that TRIM28, rather than CD133, functions as a central regulator of CSC-associated phenotypes in colorectal cancer. These findings highlight the importance of epigenetic context in CSC biology and may inform the development of more effective therapeutic strategies.