title: Side population in human non-muscle invasive bladder cancer enriches for cancer stem cells that are maintained by MAPK signalling.
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 (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.
authors.affiliation: Northern Institute for Cancer Research, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom.
authors.forename: Anastasia C
authors.initials: AC
authors.lastname: Hepburn
authors.forename: Rajan
authors.initials: R
authors.lastname: Veeratterapillay
authors.forename: Stuart C
authors.initials: SC
authors.lastname: Williamson
authors.forename: Amira
authors.initials: A
authors.lastname: El-Sherif
authors.forename: Neha
authors.initials: N
authors.lastname: Sahay
authors.forename: Huw D
authors.initials: HD
authors.lastname: Thomas
authors.forename: Alejandra
authors.initials: A
authors.lastname: Mantilla
authors.forename: Robert S
authors.initials: RS
authors.lastname: Pickard
authors.forename: Craig N
authors.initials: CN
authors.lastname: Robson
authors.forename: Rakesh
authors.initials: R
authors.lastname: Heer
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D018572:Disease-Free Survival; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020935:MAP Kinase Signaling System; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D060787:Neoplasm Grading; D009361:Neoplasm Invasiveness; D009363:Neoplasm Proteins / Q000235:genetics; D009367:Neoplasm Staging; D014411:Neoplastic Stem Cells / Q000473:pathology*; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D012008:Recurrence; D058985:Side-Population Cells / Q000473:pathology*; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology*
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3