MOLECULAR FEATURES OF TRIPLE NEGATIVE BREAST CANCER STEM CELLS: A GENE EXPRESSION PROFILING ANALYSIS OF MDA-MB-231 CELLS

Abstract

Triple negative breast cancer (TNBC) is a chemoresistant subtype of female breast tumors. Chemoresistance is attributed to the presence of long-lived cancer stem cells (CSCs) responsible for therapy failure. This study investigated the differential gene expression of signaling pathways controlling TNBC CSCs. We determined the mRNA and protein expressions of genes responsible for stemness markers, autophagy, apoptosis, CYP450 enzymes, and tumor suppressors TNBC and CSCs. We measured the caspases by fluorescence intensity and quantified cells in LC3 activated cells, reactive oxygen species (ROS), and DNA damaged cells. CSCs were identified by Aldeflour, side population, and CD44+/CD24- assay. We conducted RNA isolation using TRIZOL and measured the induction by RT-PCR for stemness markers, autophagy, apoptosis, CYP450, and tumor suppressor genes. Proteins were extracted by RIPA and quantitated by Western blot. The caspases fluorescence was detected by immunofluorescence assay. The activated cells in autophagy, oxidative stress, and DNA damage assays were explored by Muse Cell Analyzer. Independent t-test was used to detect statistical significance. The apoptotic markers BAX, caspase3, caspase8, and caspase9 were downregulated by 53%, 30%, 50%, and 50%, respectively. The cellular content of caspases was diminished in CSCs. However, the anti-apoptotic Bcl-xL was 14% higher in CSC than TNBC. The autophagy p62 gene was 61% upregulated, whereas the ATG and LC3-activated cells were lower by 33% at the protein level and 78.3% by flowcytometry, respectively. Nonetheless, CYP3A4, and CYP2D6 were lower in CSCs by 75%, 23%, 43%, 23.8%, and 17%, respectively. Moreover, CSCs were localized in G0/G1-phase. Similarly, the tumor suppressor genes BRCA (40%), PTEN (25%), and p53 (36%) were downregulated. The factors, AKT increased by 33%, NF-KB and ki-67 were decreased by 32% at the protein level, and 82.8% at the mRNA level, respectively in CSCs. Finally, CSCs expressed a 100% higher ROS+ than TNBC. TNBC CSCs are quiescent, with highly functional DNA damage repair mechanism. CSCs induce chemoresistance through downregulating genes responsible for apoptosis, autophagy, and tumor suppression. Targeting dysregulated CSCs pathways could be a potential therapy for overcoming chemoresistance.