Role of cancer stem cells in breast and prostate cancer
Cancer stem cells (CSC) are thought to be responsible for the initiation, propagation and tumour re-occurrence. CSC have been identified in most solid and haematological cancers and account for ~1% of the total cell population. Culturing cancer cell lines in monolayers enriches for the most dominant subpopulation which in most cases does not represent the slow dividing CSC population. We investigated the expression of CSC markers in 2D vs. 3D cell culture with the aim of identifying CSC-like cells via a nanog-reporter cell line and enable the subsequent targeting of these cells with CSC-targeting agents. SUM159 and MCF7 cell lines cultured in 3D cell culture significantly enriched for the CD44+/CD24- breast cancer stem cell phenotype when compared against 2D cell culture (p<0.05 and 0.001 respectively) and also enriched for expression of CD181 (p<0.05 and 0.001 respectively). However, this method did not enrich for the prostate CSC with the CD44+/CD133+ phenotype in PC3, DU145 and LNCAP cells. Using reporter cell lines to further identify the CSC population in SUM159 cell line that express GFP in response to Nanog (NRE-GFP), found that these cells were GFP+ve in the absence of Nanog protein. As these reporters were selected based on GFP that was supposedly Nanog driven other mechanistic studies were examined to determine how GFP is expressed in the NRE-GFP and control cell line. It was found that GFP could be induced in apoptosis, CSC enriching medium, hypoxia and by inhibiting the proteasome in the absence of Nanog protein. It was concluded that reporter cell lines that respond to a response element may not identify the CSC population as other factors can induce GFP expression. Compounds related to Withaferin A have been proposed to specifically target CSCs. Prostate and breast cancer cell lines cultured in 2D and 3D were treated with a novel withanolide derivative (LG-02) and Withanilode E to determine if these compounds were more effective at inducing cell cycle arrest and apoptosis using flow cytometry and microscopy. It was determined that LG-02 and WE primary mode of action is cell cycle inhibition and both compounds are more potent cell cycle inhibitors than Withaferin A. G1 phase accumulation was observed in the SUM159, PC3 and LNCAP cell lines and G2/M phase accumulation in the DU145 cell line. Cell cycle arrest was inconclusive in the MCF7 cell line. An apoptotic morphology was only significantly induced at higher concentrations in MCF7, PC3, DU145 and LNCAP. Withanolide derivatives also target the androgen response pathway, demonstrated by a decrease in PSA and androgen receptor in prostate cancer cell lines. LG-02 slowed growth of breast cancer cell lines cultured in 3D and inhibited spheroid formation of prostate cancer cell lines, however the androgen-dependent cell line LNCAP was consistently able to form 3D colonies, most likely via pAkt activation. We conclude that 3D cell culture does enrich for the CSC population in breast cancer cell lines but using a reporter cell line expressing GFP under the control of a NRE is not a suitable model for identifying the CSC population for subsequent drug treatment. In addition, Withanolide derivatives have potential anti-tumour activity and may represent a novel class of anti-androgenic agents.
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