Side population in human non-muscle invasive bladder cancer enriches for cancer stem cells that are maintained by MAPK signalling.

Anastasia C Hepburn,Alejandra Mantilla,Rajan Veeratterapillay,Neha Sahay,Amira El-Sherif,Robert S Pickard,Rakesh Heer,Huw D Thomas,Craig N Robson,Stuart C Williamson,Dean G Tang

doi:10.1371/journal.pone.0050690

Anastasia C Hepburn, Alejandra Mantilla + Show 9 more

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https://doi.org/10.1371/journal.pone.0050690

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Abstract

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 (ABCG2hi) relative to the non-SP (NSP) fraction (ABCG2low). ABCG2hi 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 ABCG2low NSP cells. In vivo, ABCG2hi SP cells enriched for tumour growth compared with ABCG2low NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2hi SP cells and MEK inhibition also inhibited the ABCG2hi 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 ABCG2hi SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.

Highlights

Transitional cell carcinoma (TCC) of the urinary bladder has a worldwide annual incidence of over 350,000 new cases and 145,000 deaths [1]
As we have shown that ABCG2hi Side population (SP) cells can give rise to both SP and NSP cells (Figure S7F) and ABCG2low NSP cells have very low levels of pERK, the suppression of ABCG2low NSP cell colony forming efficiency (CFE) appears to be indirect through the effects of U0126 on ABCG2hi SP cells
In search of a cancer stem cells (CSCs) hierarchy in human bladder cancer, our in vitro studies showed ABCG2hi SP cells displayed a three-fold increase in colony forming efficiency

Summary

Introduction

Transitional cell carcinoma (TCC) of the urinary bladder has a worldwide annual incidence of over 350,000 new cases and 145,000 deaths [1]. 85% of patients present with less aggressive non-muscle invasive bladder cancer (NMIBC), they have a high risk of recurrence and those with unfavourable characteristics such as multifocal disease, high grade tumours, with or without concomitant carcinoma in situ (CIS), are at particular risk of disease progression. For patients that present with, or progress to, muscle invasive bladder cancer (MIBC), the chance of surviving more than 5 years following treatment is approximately 50–60% [5,6]. This background indicates that novel therapeutic approaches against NMIBC are urgently needed to eradicate disease before an invasive phenotype develops [7]

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