Shireen Hourani (College of Pharmacy)| Sabah Akhtar (College of Pharmacy)| Hanaa Mousa (College of Medicine, QU Health, Qatar University)| Asad Zeidan (College of Medicine, QU Health, Qatar University)| Abdelali Agouni (College of Pharmacy)| Hesham Korashy (College of Pharmacy)
Triple negative breast cancer (TNBC) is the most aggressive and chemo-resistant subtype of breast tumors in females. This chemoresistance has been attributed to the presence of long-lived cancer stem cells (CSCs) responsible for tumor initiation and cancer self-renewal capability. However, there is not much information is known about how CSCs are different from cancer cells in terms of the expression and function of certain molecular pathways such as autophagy, apoptosis, tumor suppression, and cytochrome P450 enzymes (CYPs). Therefore, the main objective of the present study was to explore the differential gene expression and function of the signaling pathways controlling the development of triple negative breast cancer stem cells. To verify our objective, human TNBC MDA-MB-231 cells were utilized as a study model. CSCs were isolated by mammospheres formation and then identified and characterized by ALDH assay, side population assay, and CD44+/CD24- expression. The mRNA and protein expression levels of apoptotic genes (caspase-3, -7, -8, -9, and BAX), tumor suppressor genes (p53, BRCA, and PTEN), autophagy (LC3II, p62, and ATG), and CYPs (1A1, 3A4, and 2D6) were quantified by RT-PCR and Western blot analyses. In addition, changes of the apoptosis genes were further validated by detecting the cellular content and localization of caspases by immunofluorescence (IF). The independent t-test was used to detect the statistical significance (P value ≤ 0.05). Our results demonstrated that mammospheres expressed higher levels of stemness markers as compared to MDA-MB-231 cells. CSCs expressed significant downregulation of the proapoptotic genes; caspases and BAX compared to non-CSCs MDA-MB-231 at the mRNA, protein, and IF levels. Similarly, the expression of tumor suppressor genes; p53, BRCA, and PTEN was significantly lower in CSCs than in MDA-MB-231 cells. In addition, the autophagy p62 protein level was higher in CSCs than in MA-MB-231 cells. With regards to drug metabolizing enzymes, CSCs expressed higher CYP1A1 mRNA and protein expression levels than MDA-MB-231 cells, whereas the protein expression levels of CYP3A4 and CYP2D6 were downregulated in CSCs in comparison to MDA-MB-231 cells. In conclusion, our results suggest that the triple negative breast cancer stem cells’ chemoresistance and is mainly attributed to impaired expression of tumour suppressor and pro-apoptotic genes, which make CSCs resistance to apoptosis and thus maintain their development and proliferation. This study sheds the light on the importance of targeting the dysregulated apoptotic pathway of cancer stem cells to overcome chemoresistance and preventing tumor relapse.
Support or Funding Information
The Office Research Support at Qatar University, Qatar (grants # IRCC-2019-006 and QUCG-CPH-20/21-1) and the Qatar National Research Fund, Qatar Foundation, Qatar (grant # UREP24-163-3-049 and UREP27-160-3-045).