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Cancer stem cells (CSCs) represent rare tumor cell populations capable of self-renewal, differentiation, and tumor initiation and are highly resistant to chemotherapy and radiotherapy. Thus, therapeutic approaches that can effectively target CSCs and tumor cells could be the key to efficient tumor treatment. In this study, we explored the function of SPHK1 in breast CSCs and non-CSCs. We showed that RNAi-mediated knockdown of SPHK1 inhibited cell proliferation and induced apoptosis in both breast CSCs and non-CSCs, while ectopic expression of SPHK1 enhanced breast CSC survival and mammosphere forming efficiency. We identified STAT1 and IFN signaling as key regulatory targets of SPHK1 and demonstrated that an important mechanism by which SPHK1 promotes cancer cell survival is through the suppression of STAT1. We further demonstrated that SPHK1 inhibitors, FTY720 and PF543, synergized with doxorubicin in targeting both breast CSCs and non-CSCs. In conclusion, we provide important evidence that SPHK1 is a key regulator of cell survival and proliferation in breast CSCs and non-CSCs and is an attractive target for the design of future therapies.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent due to its selective killing on cancer cells while sparing the normal cells. Nevertheless, breast adenocarcinoma cells can develop TRAIL resistance. Therefore, this project investigated the anti-cancer effects of the combination of epigenetic drugs zebularine and trichostatin A (ZT) with TRAIL (TZT) on the human breast adenocarcinoma cells. This treatment regimen was compared with the natural anti-cancer compound curcumin (Cur) and standard chemotherapeutic drug doxorubicin (Dox). As compared to TRAIL treatment, TZT treatment hampered the cell viability of human breast adenocarcinoma cells MDA-MB-231 significantly but not MCF-7 and immortalized non-cancerous human breast epithelial cells MCF10A. Unlike TZT, Cur and Dox treatments reduced cell viability in both human breast adenocarcinoma and epithelial cells significantly. Nevertheless, there were no changes in cell cycle in both TRAIL and TZT treatments in breast adenocarcinoma and normal epithelial cells. Intriguingly, Cur and Dox treatment generally induced G2/M arrest in MDA-MB-231, MCF-7 and MCF10A but Cur induced S phase arrest in MCF10A. The features of apoptosis such as morphological changes, apoptotic activity and the expression of cleaved poly (ADP) ribose polymerase (PARP) protein were more prominent in TRAIL and TZT-treated MDA-MB-231 as compared to MCF10A at 24 h post-treatment. Compared to TZT treatment, Cur and Dox treatments exhibited lesser apoptotic features in MDA-MB-231. Collectively, the sensitization using Zeb and TSA to augment TRAIL-induced apoptosis might be an alternative therapy towards human breast adenocarcinoma cells, without harming the normal human breast epithelial cells.

Background: Mutations in p53 gene are observed in ∼50% of all human cancers. In breast cancer alone, 12%-32% of luminal, 84% of basal-like and 75% of HER-2 expressing tumors have apparent p53 mutations. Tumor cells undergo epithelial-mesenchymal transition (EMT) to metastasise from primary sites to form secondary tumors at distant regions of the body. EMT is a complex biologic phenomenon which governs the transition of cancer cells with epithelial characteristics to mesenchymal traits, gaining new properties such as aggressiveness and invasiveness. Recent studies revealed that mutations in the p53 gene can give rise to alternate functional phenotypes leading to tumor initiation and progression. Aim: The aim of this study is to develop a robust human breast cancer cellular model to investigate p53 gain-of-function (GOF) mutations and EMT as well as evaluating the EMT phenotype associated with these mutations. Methods: Two breast cancer cell lines, namely MDA-MB-468 and HCC38 carrying the R273H and R273L missense mutations, respectively, were subjected to p53 knockdown using shRNA directed against p53 gene through lentiviral vector transduction. The transduced cells were then harvested for Western blotting to evaluate the protein expression of EMT markers which includes E-cadherin, SNAIL, ZEB1 and vimentin compared with the nontransduced control cells. Subsequently, both cell lines were subjected to mammosphere generation and redifferentiation to determine the basal expression of the EMT markers. Results: Silencing of p53 using siRNA in MDA-MB-468 and HCC38 downregulated E-cadherin expressions but upregulated vimentin levels. Furthermore, E-cadherin levels reduced significantly after conversion from adherent parental cells to mammospheres, but rebound upon redifferentiation. Conversely, vimentin was upregulated in mammospheres as compared with the parental and redifferentiated groups. Conclusion: p53 knockdown in breast cancer cells harboring R273H and R273L mutations favor vimentin expression but not E-cadherin, suggesting that p53-GOF mutants are involved in EMT and the development of metastatic tumors. The mammosphere model accurately recapitulates cell plasticity between epithelial and mesenchymal states, as evidenced by the expression of mesenchymal cell markers in the mammospheres, and epithelial cell markers in adherent and redifferentiated cells.