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High-throughput flow cytometry screening reveals a role for junctional adhesion molecule a as a cancer stem cell maintenance factor.

ABSTRACT: Stem cells reside in niches that regulate the balance between self-renewal and differentiation. The identity of a stem cell is linked with the ability to interact with its niche through adhesion mechanisms. To identify targets that disrupt cancer stem cell (CSC) adhesion, we performed a flow cytometry screen on patient-derived glioblastoma (GBM) cells and identified junctional adhesion molecule A (JAM-A) as a CSC adhesion mechanism essential for self-renewal and tumor growth. JAM-A was dispensable for normal neural stem/progenitor cell (NPC) function, and JAM-A expression was reduced in normal brain versus GBM. Targeting JAM-A compromised the self-renewal of CSCs. JAM-A expression negatively correlated to GBM patient prognosis. Our results demonstrate that GBM-targeting strategies can be identified through screening adhesion receptors and JAM-A represents a mechanism for niche-driven CSC maintenance.

SUBMITTER: Lathia JD

PROVIDER: S-EPMC3899718 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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High-throughput flow cytometry screening reveals a role for junctional adhesion molecule a as a cancer stem cell maintenance factor.

Lathia Justin D JD Li Meizhang M Sinyuk Maksim M Alvarado Alvaro G AG Flavahan William A WA Stoltz Kevin K Rosager Ann Mari AM Hale James J Hitomi Masahiro M Gallagher Joseph J Wu Qiulian Q Martin Jody J Vidal Jason G JG Nakano Ichiro I Dahlrot Rikke H RH Hansen Steinbjørn S McLendon Roger E RE Sloan Andrew E AE Bao Shideng S Hjelmeland Anita B AB Carson Christian T CT Naik Ulhas P UP Kristensen Bjarne B Rich Jeremy N JN

Cell reports 20131227 1

Stem cells reside in niches that regulate the balance between self-renewal and differentiation. The identity of a stem cell is linked with the ability to interact with its niche through adhesion mechanisms. To identify targets that disrupt cancer stem cell (CSC) adhesion, we performed a flow cytometry screen on patient-derived glioblastoma (GBM) cells and identified junctional adhesion molecule A (JAM-A) as a CSC adhesion mechanism essential for self-renewal and tumor growth. JAM-A was dispensab ...[more]

PMID: 24373972

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Project description:The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen-sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen-sensitive tissues. A variety of reporter systems have been developed, driven by androgen-sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high-throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry. Androgen-independent human prostate cancer-derived PC3 cells were transiently cotransfected with an expression vector for the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was coadministered at submaximal concentrations with potential AR antagonists. The assay was formatted for high-throughput screening using the HyperCyt flow cytometry system. Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose-dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when coadministered with low-dose R1881, which also occurred in a dose-dependent manner. Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high-throughput format. Dose-response curves can be successfully generated for these compounds, allowing for an assessment of potency. Because of its simplicity and high-throughput compatibility, the MARS assay and HyperCyt system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.

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High-throughput flow cytometry screening reveals a role for junctional adhesion molecule a as a cancer stem cell maintenance factor.

Publications

High-throughput flow cytometry screening reveals a role for junctional adhesion molecule a as a cancer stem cell maintenance factor.

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