Search

Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. In vitro and in vivo phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2(hi)) relative to the non-SP (NSP) fraction (ABCG2(low)). ABCG2(hi) SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2(low) NSP cells. In vivo, ABCG2(hi) SP cells enriched for tumour growth compared with ABCG2(low) NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2(hi) SP cells and MEK inhibition also inhibited the ABCG2(hi) SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2(hi) SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.

Stem cells are thought to be the cell of origin in malignant transformation in many tissues, but their role in human prostate carcinogenesis continues to be debated. One of the conflicts with this model is that cancer stem cells have been described to lack androgen receptor (AR) expression, which is of established importance in prostate cancer initiation and progression. We re-examined the expression patterns of AR within adult prostate epithelial differentiation using an optimised sensitive and specific approach examining transcript, protein and AR regulated gene expression. Highly enriched populations were isolated consisting of stem (α(2)β(1)(HI) CD133(+VE)), transiently amplifying (α(2)β(1)(HI) CD133(-VE)) and terminally differentiated (α(2)β(1)(LOW) CD133(-VE)) cells. AR transcript and protein expression was confirmed in α(2)β(1)(HI) CD133(+VE) and CD133(-VE) progenitor cells. Flow cytometry confirmed that median (±SD) fraction of cells expressing AR were 77% (±6%) in α(2)β(1)(HI) CD133(+VE) stem cells and 68% (±12%) in α(2)β(1)(HI) CD133(-VE) transiently amplifying cells. However, 3-fold lower levels of total AR protein expression (peak and median immunofluorescence) were present in α(2)β(1)(HI) CD133(+VE) stem cells compared with differentiated cells. This finding was confirmed with dual immunostaining of prostate sections for AR and CD133, which again demonstrated low levels of AR within basal CD133(+VE) cells. Activity of the AR was confirmed in prostate progenitor cells by the expression of low levels of the AR regulated genes PSA, KLK2 and TMPRSS2. The confirmation of AR expression in prostate progenitor cells allows integration of the cancer stem cell theory with the established models of prostate cancer initiation based on a functional AR. Further study of specific AR functions in prostate stem and differentiated cells may highlight novel mechanisms of prostate homeostasis and insights into tumourigenesis.