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Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo.

ABSTRACT: Nicastrin (NCT) is a crucial component of the ?-secretase (GS) enzyme, which prompted investigations into its biological role in cancer. We have previously shown that nicastrin is overexpressed in breast cancer (BC), conferring worse overall survival in invasive, ER? negative patients. Here, we used 2D and 3D Matrigel, anchorage-independent growth conditions and a breast cancer xenograft mouse model to assess the impact of nicastrin on breast cancer stem cell (BCSC) propagation and invasion in vitro and tumor growth in vivo. Stable knockdown of nicastrin in HCC1806 breast cancer cells reduced cell invasion by 51.4 ± 1.7%, accompanied by a morphological change to a rounded cell phenotype and down-regulation of vimentin, Snail, Twist, MMP2, and MMP9. We observed a reduction of the pool of CD44(+)/CD24(-) and ALDH1 high breast cancer stem cells by threefold and twofold, respectively, and a reduction by 2.6-fold of the mammospheres formation. Nicastrin overexpression in nontransformed MCF10A cells caused an induction of epithelial to mesenchymal regulators, as well as a fivefold increased ALDH1 activity, a threefold enrichment for CD44(+)/CD24(-) stem cells, and a 3.2-fold enhanced mammosphere-forming capacity. Using the ?-sescretase inhibiton, Notch1/4 siRNA, and Akt inhibition, we show that nicastrin regulates breast cancer stem cells partly through Notch1 and the Akt pathway. Exploiting serial dilution transplantation of the HCC1806 cells expressing nicastrin and HCC1806 stably depleted of nicastrin, in vivo, we demonstrate that nicastrin inhibition may be relevant for the reduced tumorigenicity of breast cancer cells. These data could serve as a benchmark for development of nicastrin-targeted therapies in breast cancer.

SUBMITTER: Lombardo Y

PROVIDER: S-EPMC3478621 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo.

Lombardo Ylenia Y Filipović Aleksandra A Molyneux Gemma G Periyasamy Manikandan M Giamas Georgios G Hu Yunhui Y Trivedi Pritesh S PS Wang Jayson J Yagüe Ernesto E Michel Loren L Coombes R Charles RC

Proceedings of the National Academy of Sciences of the United States of America 20120924 41

Nicastrin (NCT) is a crucial component of the γ-secretase (GS) enzyme, which prompted investigations into its biological role in cancer. We have previously shown that nicastrin is overexpressed in breast cancer (BC), conferring worse overall survival in invasive, ERα negative patients. Here, we used 2D and 3D Matrigel, anchorage-independent growth conditions and a breast cancer xenograft mouse model to assess the impact of nicastrin on breast cancer stem cell (BCSC) propagation and invasion in v ...[more]

PMID: 23012411

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Project description:BackgroundCancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines.MethodsSpheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo.ResultsCircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth.ConclusionsCircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.

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Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo.

Publications

Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo.

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