Epigenetic Regulations of Cancer Stem Cells by the Aryl Hydrocarbon Receptor Pathway

Compelling evidence has demonstrated that tumor bulk comprises distinctive subset of cells generally referred as cancer stem cells (CSCs) that has been proposed as a strong sustainer and promoter of tumorigenesis and therapeutic resistance. These distinguished properties of CSCs have raised interest in understanding the molecular mechanisms that govern the maintenance of these cells. Numerous experimental and epidemiological studies have demonstrated that exposure to environmental toxins such as the polycyclic aromatic hydrocarbons (PAHs) is strongly involved in cancer initiation and progression. The PAH-induced carcinogenesis is shown to be mediated through the activation of a cytosolic receptor, aryl hydrocarbon receptor (AhR)/Cytochrome P4501A pathway, suggesting a possible direct link between AhR and CSCs. Several recent studies have investigated the role of AhR in CSCs self-renewal and maintenance, however the molecular mechanisms and particularly the epigenetic regulations of CSCs by AhR have not been reviewed before. In this review, we first summarize the crosstalk between AhR and cancer genetics, with particular emphasis on mechanisms relevant to CSCs such as Wnt/β-catenin pathway, Notch pathway, NF-κB pathway, PTEN-PI3K/Akt pathway and Drug Resistance-mediating pathways. The second part of this review discusses the recent advances and studies highlighting the epigenetic mechanisms mediated by the AhR pathway that control CSC gene expression, self-renewal, and chemoresistance in various human cancers. Furthermore, the review also sheds light on the importance of targeting the epigenetic pathways as a novel therapeutic approach against CSCs

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

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

EGFR Inhibitor Gefitinib Induces Cardiotoxicity through the Modulation of Cardiac PTEN/Akt/FoxO3a Pathway and Reactive Metabolites Formation: and Rat Studies.

Gefitinib (GEF) is a selective inhibitor of the epidermal growth factor receptor (EGFR) used to treat non-small cell lung cancer. Yet, few cases of cardiotoxicity have been reported. However, the role of the PTEN/Akt/FoxO3a pathway, which mediates GEF anticancer activity, in GEF cardiotoxicity remains unclear. For this purpose, H9c2 cells and rat cardiomyocytes were utilized as study models. Treatment of H9c2 cells and Sprague-Dawley rats with GEF significantly induced the expression of hypertrophic and apoptotic markers at mRNA and protein levels with an increased plasma level of troponin. This was accompanied by induction of autophagy and mitochondrial dysfunction in H9c2 cells. Inhibition of cardiac EGFR activity and Akt cellular content of and rat cardiomyocytes by GEF increased PTEN and FoxO3a gene expression and cellular content. Importantly, treatment of H9c2 cells with PI3K/Akt inhibitor increased PTEN and FoxO3a mRNA expression associated with potentiation of GEF cardiotoxicity. In addition, by using LC-MS/MS, we showed that GEF is metabolized in the rat heart microsomes into one cyanide- and two methoxylamine-adduct reactive metabolites, where their formation was entirely blocked by CYP1A1 inhibitor, α-naphthoflavone. The current study concludes that GEF induces cardiotoxicity through modulating the expression and function of the cardiac PTEN/AKT/FoxO3a pathway and the formation of CYP1A1-mediated reactive metabolites