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Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator ΔNp63α.

Portillo-Lara R, Alvarez MM - PLoS ONE (2015)

Bottom Line: Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures.We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology.This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Monterrey, Nuevo León, México.

ABSTRACT

Background: Cancer stem cells (CSC) drive prostate cancer tumor survival and metastasis. Nevertheless, the development of specific therapies against CSCs is hindered by the scarcity of these cells in prostate tissues. Suspension culture systems have been reported to enrich CSCs in primary cultures and cell lines. However, the molecular mechanisms underlying this phenomenon have not been fully explored.

Methodology/principal findings: We describe a prostasphere assay for the enrichment of CD133+ CSCs in four commercial PCa cell lines: 22Rv1, DU145, LNCaP, and PC3. Overexpression of CD133, as determined by flow cytometric analysis, correlated with an increased clonogenic, chemoresistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Gene expression profiling was then carried out using the Cancer Reference panel and the nCounter system from NanoString Technologies. This analysis revealed several upregulated transcripts that can be further explored as potential diagnostic markers or therapeutic targets. Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures.

Conclusions/significance: We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology. This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

No MeSH data available.


Related in: MedlinePlus

Gene expression profiling and functional annotation analysis: Activation of developmental pathways modulated by ΔNp63α drives the CSC-enrichment observed in prostasphere cultures.A) Heat map showing fold changes in gene expression from prostaspheres at day 4 (P2), day 8 (P3), day 12 (P4), and isolated CD133- (P4_N) and CD133+ (P4_P) fractions relative to parental cultures on day 0. Values correspond to the Log2 transformed ratios of the means (n = 3). The figure shows the behavior of the 244 genes assayed throughout the time the cells are kept in culture. B) Gene Ontology (GO) analysis of upregulated transcripts in CD133+ cells. GO Biological processes enriched in CD133+ cells are all related to developmental pathways (p* < 0.01). The edge width reflects the relative overlap between the nodes (% shared genes). The edge color encodes the number of shared members between the whole GO category and the user-defined background (CodeSets). Sphere size is relative to the number of genes associated with that category. C) Molecular interactions of the transcriptional regulator ΔNp63α with upregulated genes detected in CD133+ cells. ATM, TP63, IGFBP3, NOTCH1, BRCA2, IL1A and TOP2 genes are upregulated in CD133+ cells and appear to mediate the downstream activation of developmental molecular pathways. The ΔNp63α tetramer shown at the center of the figure is known to physically interact with these genes. Pathway source: Pathway interaction database (http://pid.nci.nih.gov).
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pone.0130118.g006: Gene expression profiling and functional annotation analysis: Activation of developmental pathways modulated by ΔNp63α drives the CSC-enrichment observed in prostasphere cultures.A) Heat map showing fold changes in gene expression from prostaspheres at day 4 (P2), day 8 (P3), day 12 (P4), and isolated CD133- (P4_N) and CD133+ (P4_P) fractions relative to parental cultures on day 0. Values correspond to the Log2 transformed ratios of the means (n = 3). The figure shows the behavior of the 244 genes assayed throughout the time the cells are kept in culture. B) Gene Ontology (GO) analysis of upregulated transcripts in CD133+ cells. GO Biological processes enriched in CD133+ cells are all related to developmental pathways (p* < 0.01). The edge width reflects the relative overlap between the nodes (% shared genes). The edge color encodes the number of shared members between the whole GO category and the user-defined background (CodeSets). Sphere size is relative to the number of genes associated with that category. C) Molecular interactions of the transcriptional regulator ΔNp63α with upregulated genes detected in CD133+ cells. ATM, TP63, IGFBP3, NOTCH1, BRCA2, IL1A and TOP2 genes are upregulated in CD133+ cells and appear to mediate the downstream activation of developmental molecular pathways. The ΔNp63α tetramer shown at the center of the figure is known to physically interact with these genes. Pathway source: Pathway interaction database (http://pid.nci.nih.gov).

Mentions: Gene expression profiling of cancer tissues has the potential to improve current diagnostic and prognostic classifications, and to reveal insight into the underlying CSC pathophysiology. Here, we aimed to identify overexpressed genes in magnetically sorted and unsorted PC3 prostasphere cells, compared to parental monolayer cultures. Gene expression data was submitted to the Gene Expression Omnibus repository from NCBI and can be accessed at the GEO website (http://www.ncbi.nlm.nih.gov/geo/) under the GSE67248 accession number. Table 1 lists upregulated transcripts (ratio > 1.5) in CD133+ sorted and unsorted prostasphere cells (See Table A in S1 File for the full set of data). Fig 6A shows the hierarchical clustering of the relative fold changes in the 244 genes analyzed, throughout the 12 days of culture. This graphical representation shows how culture conditions induce global changes in gene expression through time. Functional annotation clustering of upregulated genes with similar biological function included: proteins containing predicted glycosylation sites and sequences targeting them to the secretory pathway, membrane-associated proteins, proteins that participate in cell adhesion and cell surface structure organization, and proteins with tyrosine kinase activity.


Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator ΔNp63α.

Portillo-Lara R, Alvarez MM - PLoS ONE (2015)

Gene expression profiling and functional annotation analysis: Activation of developmental pathways modulated by ΔNp63α drives the CSC-enrichment observed in prostasphere cultures.A) Heat map showing fold changes in gene expression from prostaspheres at day 4 (P2), day 8 (P3), day 12 (P4), and isolated CD133- (P4_N) and CD133+ (P4_P) fractions relative to parental cultures on day 0. Values correspond to the Log2 transformed ratios of the means (n = 3). The figure shows the behavior of the 244 genes assayed throughout the time the cells are kept in culture. B) Gene Ontology (GO) analysis of upregulated transcripts in CD133+ cells. GO Biological processes enriched in CD133+ cells are all related to developmental pathways (p* < 0.01). The edge width reflects the relative overlap between the nodes (% shared genes). The edge color encodes the number of shared members between the whole GO category and the user-defined background (CodeSets). Sphere size is relative to the number of genes associated with that category. C) Molecular interactions of the transcriptional regulator ΔNp63α with upregulated genes detected in CD133+ cells. ATM, TP63, IGFBP3, NOTCH1, BRCA2, IL1A and TOP2 genes are upregulated in CD133+ cells and appear to mediate the downstream activation of developmental molecular pathways. The ΔNp63α tetramer shown at the center of the figure is known to physically interact with these genes. Pathway source: Pathway interaction database (http://pid.nci.nih.gov).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4481544&req=5

pone.0130118.g006: Gene expression profiling and functional annotation analysis: Activation of developmental pathways modulated by ΔNp63α drives the CSC-enrichment observed in prostasphere cultures.A) Heat map showing fold changes in gene expression from prostaspheres at day 4 (P2), day 8 (P3), day 12 (P4), and isolated CD133- (P4_N) and CD133+ (P4_P) fractions relative to parental cultures on day 0. Values correspond to the Log2 transformed ratios of the means (n = 3). The figure shows the behavior of the 244 genes assayed throughout the time the cells are kept in culture. B) Gene Ontology (GO) analysis of upregulated transcripts in CD133+ cells. GO Biological processes enriched in CD133+ cells are all related to developmental pathways (p* < 0.01). The edge width reflects the relative overlap between the nodes (% shared genes). The edge color encodes the number of shared members between the whole GO category and the user-defined background (CodeSets). Sphere size is relative to the number of genes associated with that category. C) Molecular interactions of the transcriptional regulator ΔNp63α with upregulated genes detected in CD133+ cells. ATM, TP63, IGFBP3, NOTCH1, BRCA2, IL1A and TOP2 genes are upregulated in CD133+ cells and appear to mediate the downstream activation of developmental molecular pathways. The ΔNp63α tetramer shown at the center of the figure is known to physically interact with these genes. Pathway source: Pathway interaction database (http://pid.nci.nih.gov).
Mentions: Gene expression profiling of cancer tissues has the potential to improve current diagnostic and prognostic classifications, and to reveal insight into the underlying CSC pathophysiology. Here, we aimed to identify overexpressed genes in magnetically sorted and unsorted PC3 prostasphere cells, compared to parental monolayer cultures. Gene expression data was submitted to the Gene Expression Omnibus repository from NCBI and can be accessed at the GEO website (http://www.ncbi.nlm.nih.gov/geo/) under the GSE67248 accession number. Table 1 lists upregulated transcripts (ratio > 1.5) in CD133+ sorted and unsorted prostasphere cells (See Table A in S1 File for the full set of data). Fig 6A shows the hierarchical clustering of the relative fold changes in the 244 genes analyzed, throughout the 12 days of culture. This graphical representation shows how culture conditions induce global changes in gene expression through time. Functional annotation clustering of upregulated genes with similar biological function included: proteins containing predicted glycosylation sites and sequences targeting them to the secretory pathway, membrane-associated proteins, proteins that participate in cell adhesion and cell surface structure organization, and proteins with tyrosine kinase activity.

Bottom Line: Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures.We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology.This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Monterrey, Nuevo León, México.

ABSTRACT

Background: Cancer stem cells (CSC) drive prostate cancer tumor survival and metastasis. Nevertheless, the development of specific therapies against CSCs is hindered by the scarcity of these cells in prostate tissues. Suspension culture systems have been reported to enrich CSCs in primary cultures and cell lines. However, the molecular mechanisms underlying this phenomenon have not been fully explored.

Methodology/principal findings: We describe a prostasphere assay for the enrichment of CD133+ CSCs in four commercial PCa cell lines: 22Rv1, DU145, LNCaP, and PC3. Overexpression of CD133, as determined by flow cytometric analysis, correlated with an increased clonogenic, chemoresistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Gene expression profiling was then carried out using the Cancer Reference panel and the nCounter system from NanoString Technologies. This analysis revealed several upregulated transcripts that can be further explored as potential diagnostic markers or therapeutic targets. Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures.

Conclusions/significance: We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology. This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

No MeSH data available.


Related in: MedlinePlus