APC mutation is the main driving mechanism of CRC development and leads to constitutively activated WNT signaling, overpopulation of ALDH+ stem cells (SCs), and incomplete differentiation. We previously reported that retinoic acid (RA) receptors are selectively expressed in ALDH+ SCs, which provides a way to target cancer SCs with retinoids to induce differentiation. Hypotheses: A functional link exists between the WNT and RA pathways, and APC mutation generates a WNT:RA imbalance that decreases retinoid-induced differentiation and increases ALDH+ SCs. Accordingly, to restore parity in WNT:RA signaling, we induce wt-APC expression in APC-mutant CRC cells, and we assess the ability of all-trans retinoic acid (ATRA) to induce differentiation. We found that ATRA increased expression of the WNT target gene, CYP26A1, and inducing wt-APC reduced this expression by 50%. Thus, the RA and WNT pathways crosstalk to modulate CYP26A1, which metabolizes retinoids. Moreover, inducing wt-APC augments ATRA-induced cell differentiation by: (i) decreasing cell proliferation; (ii) suppressing ALDH1A1 expression; (iii) decreasing ALDH+ SCs; and (iv) increasing neuroendocrine cell differentiation. A novel CYP26A1-based network that links WNT and RA signaling was also identified by NanoString profiling/bioinformatics analysis. Furthermore, CYP26A1 inhibitors sensitized CRC cells to the anti-proliferative effect of drugs that downregulate WNT signaling. Notably, in wt-APC-CRCs, decreased CYP26A1 improved patient survival. These findings have strong potential for clinical translation.

The metabolic enzyme aldehyde dehydrogenase 1A1 (ALDH1A1), a cancer stem cell marker associated with poor outcomes in breast cancer, has emerged as a promising therapeutic target in TNBC. The aim of this study was to investigate the role of ALDH1A1 in radiation resistance and redox stress in triple negative breast cancer (TNBC). Functional knockouts of ALDH1A1 were generated by the CRISPR/Cas9-mediated deletion of ALDH1A1 in the SUM159 cell line, and three distinct clonal populations were isolated. Genetic targeting was confirmed by Sanger sequencing, and the loss of ALDH1A1 protein expression was validated by Western blotting. Functional assays assessed ALDEFLUOR activity, cell viability, self-renewal capacity, and reactive oxygen species (ROS) levels with or without radiation in both the bulk population and clonal lines. Interestingly, ALDEFLUOR activity was uniformly lost across all clonal lines; however, functional effects of ALDH1A1 loss on redox stress, survival, and radiation sensitivity were observed in only one clonal population. These findings highlight significant variability in the role of ALDH1A1 among clonal populations, reflecting the complexity of tumor heterogeneity. This underscores the importance of accounting for tumor heterogeneity when targeting ALDH1A1, as certain TNBC subpopulations may rely more heavily on ALDH1A1 function. These insights are critical for developing effective ALDH1A1-targeted therapies.

APC mutation is the main driver mechanism of CRC development and leads to constitutively activated WNT signaling, overpopulation of ALDH+ stem cells (SCs), and incomplete differentiation. We previously reported that retinoic acid (RA) receptors are selectively expressed in ALDH+ SCs, which provides a way to target cancer SCs with retinoids to induce differentiation. Hypotheses: A functional link exists between WNT and RA pathways, and APC mutation generates a WNT:RA imbalance that decreases retinoid-induced differentiation and increases ALDH+ SCs. Accordingly, to restore parity in WNT:RA signaling, we induced wt-APC expression in APC-mutant CRC cells and assessed the ability of all-trans-retinoic acid (ATRA) to induce differentiation. Notably, ATRA substantially increased expression of the WNT-target gene, CYP26A1, and inducing wt-APC reduced this expression by 50%. Thus, RA and WNT pathways crosstalk to modulate CYP26A1 metabolism of retinoids. We found that inducing wt-APC augments ATRA-induced cell differentiation by: i) decreasing cell proliferation; ii) suppressing ALDH1A1 expression; iii) decreasing ALDH+ SCs; iv) increasing neuroendocrine cell differentiation. Furthermore, NanoString profiling/bioinformatics identified a novel CYP26A1-based network that links WNT and RA signaling. Moreover, CYP26A1 inhibitors sensitized CRC cells to the anti-proliferative effect of drugs that downregulate WNT signaling. Notably. in wt-APC-CRCs, decreased CYP26A1 improves patient survival. These findings have strong potential for clinical translation.

The metabolic enzyme Aldehyde Dehydrogenase 1A1 (ALDH1A1), a cancer stem cell marker associated with poor outcomes in breast cancer, has emerged as a promising therapeutic target in TNBC. The aim of this study was to investigate the role of ALDH1A1 in radiation resistance and redox stress in TNBC. Functional knockouts of ALDHA1A1 were generated by CRISPR/Cas9-mediated deletion of ALDH1A1 in the SUM159 cell line, and three distinct clonal populations were isolated. Genetic targeting was confirmed by Sanger sequencing, and loss of ALDH1A1 protein expression was validated by Western blotting. Functional assays assessed ALDEFLUOR activity, cell viability, self-renewal capacity, and reactive oxygen species (ROS) levels with or without radiation in both the bulk population and clonal lines. Interestingly, ALDEFLUOR activity was uniformly lost across all clonal lines; however, functional effects of ALDH1A1 loss on redox stress, survival, and radiation sensitivity were observed in only one clonal population. These findings highlight significant variability in the role of ALDH1A1 among clonal populations, reflecting the complexity of tumor heterogeneity. This underscores the importance of accounting for tumor heterogeneity when targeting ALDH1A1, as certain TNBC subpopulations may rely more heavily on ALDH1A1 function. These insights are critical for developing effective ALDH1A1-targeted therapies.