Analysis of regulation of cell death pathway genes in CSC-enriched TNBC after treatment with doxorubicin: A potencial biomarker.

Abstract

e13154 Background: Breast cancer is one of the most common cancers in women worldwide, and triple-negative breast cancer (TNBC) accounts for approximately 10-20% of all cases globally. TNBC has the worst prognosis among other types of breast cancer due to the lack of targeted therapy. In addition, TNBC tumors exhibit significant cellular and molecular heterogeneity, along with the presence of cells with cancer stem cell (CSC) characteristics, which contribute to the low response to available chemotherapy treatments, leading to chemoresistance, recurrence and metastasis. objective of this study is to evaluate the expression of genes related to cell death and survival pathways in CSCs-enriched spheroids DOX-treated obtained of BT-549 cells, a human TNBC cell line. The death receptor genes include TNFRSF10B, TNFRSF10C, CFLAR, RIPIK1, CASP8, NFKB1, RELA, and MLKL. Methods: To achieve this, gene expression was analyzed through RT-qPCR, comparing the expression in DOX-treated CSCs-enriched spheroids (IC50 55.7 μM/ 48h incubation) and untreated CSCs-enriched spheroids. Preliminary results indicated that, among the evaluated genes, only CFLAR, NFKB1, and RELA were expressed in the untreated CSCs-enriched spheroids, while the others were not expressed under any condition. Results: The genes CFLAR, NF- κ B and RELA showed considerable Ct values, while the remaining genes did not exhibit a Ct value in one or more samples. The gene CFLAR showed an overexpression, both in monolayers vs. spheroids and in treated spheroids vs. untreated spheroids, which can be related to the inhibition of Caspase 8 and consequently, the apoptotic activity of cells. The overexpression of the RELA, same as CFLAR, suggests increased inflammation pathway activation in the tumor microenvironment. However, the lower expression of the NF-κB1 gene in treated spheroids vs. untreated spheroids may be related to the production of ROS, suppressing its activation. Conclusions: The expression of NFKB1 and RELA genes is associated with the cellular survival pathway, which may be linked to the resistance to the drug observed in many patients. Previous studies of our group have shown that achieving a satisfactory result of DOX action on CSCs-enriched spheroids requires using a dose 100 times higher than that used in monolayer cells. This may be related to the non-expression of the TNFRSF10B, TNFRSF10C, RIPIK1, CASP8, and MLKL genes in CSCs-enriched spheroid DOX-treated. Thus, the results obtained so far indicate that DOX may have an impact on the expressed genes in CSCs-enriched spheroids and demonstrate the expression of apoptosis inhibitors in treated breast spheres. Consequently, the expression of these genes in BT-549 cells TNBC indicates a pro-inflammatory, anti-apoptotic, and pro-cell survival profile of CSC-enriched spheroids.