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NF-kappa Β -inducing kinase regulates stem cell phenotype in breast cancer

View Article: PubMed Central - PubMed

ABSTRACT

Breast cancer stem cells (BCSCs) overexpress components of the Nuclear factor-kappa B (NF-κB) signaling cascade and consequently display high NF-κB activity levels. Breast cancer cell lines with high proportion of CSCs exhibit high NF-κB-inducing kinase (NIK) expression. The role of NIK in the phenotype of cancer stem cell regulation is poorly understood. Expression of NIK was analyzed by quantitative RT-PCR in BCSCs. NIK levels were manipulated through transfection of specific shRNAs or an expression vector. The effect of NIK in the cancer stem cell properties was assessed by mammosphere formation, mice xenografts and stem markers expression. BCSCs expressed higher levels of NIK and its inhibition through small hairpin (shRNA), reduced the expression of CSC markers and impaired clonogenicity and tumorigenesis. Genome-wide expression analyses suggested that NIK acts on ERK1/2 pathway to exert its activity. In addition, forced expression of NIK increased the BCSC population and enhanced breast cancer cell tumorigenicity. The in vivo relevance of these results is further supported by a tissue microarray of breast cancer samples in which we observed correlated expression of Aldehyde dehydrogenase (ALDH) and NIK protein. Our results support the essential involvement of NIK in BCSC phenotypic regulation via ERK1/2 and NF-κB.

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(A) Principal component analysis of gene expression profiles for high NIK-expressing MCF7 cells, low NIK-expressing MCF7 cells and MCF7 control cells. (B) Clustering diagram of samples and differentially expressed genes in MCF7 cells overexpressing NIK and NIK-deficient MCF7 cells. Log-Fold Changes of differentially expressed genes are depicted in a heat map, scaling from low (green) to high (red). Experiments were performed in three independent transiently-transfected MCF7 cell lines (C). Graph showing the fold change of stem cells-associated genes in high NIK-expressing cells and (D) low NIK-expressing cells. (E) RT-qPCR analysis of EGR1, TCN1, DUSP6, GDF15, CSPG4 and MAP2K6 in MCF7 overexpressing-NIK cells, MCF7 control cells, NIK-deficient MCF7 cells (shNIK1 and shNIK2) and MCF7 control cells (shLuc). All RT-qPCR were normalized to TBP. (n = 3, error bars are +/− s.d, p < 0.05). (G) IPA network of top gene networks from NIK-overexpressing cells. (H) IPA network of top gene networks from NIK-depleted cells. Note that ERK1/2 is a central node in both networks. Red color indicates induced genes and green color represents suppressed genes.
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f8: (A) Principal component analysis of gene expression profiles for high NIK-expressing MCF7 cells, low NIK-expressing MCF7 cells and MCF7 control cells. (B) Clustering diagram of samples and differentially expressed genes in MCF7 cells overexpressing NIK and NIK-deficient MCF7 cells. Log-Fold Changes of differentially expressed genes are depicted in a heat map, scaling from low (green) to high (red). Experiments were performed in three independent transiently-transfected MCF7 cell lines (C). Graph showing the fold change of stem cells-associated genes in high NIK-expressing cells and (D) low NIK-expressing cells. (E) RT-qPCR analysis of EGR1, TCN1, DUSP6, GDF15, CSPG4 and MAP2K6 in MCF7 overexpressing-NIK cells, MCF7 control cells, NIK-deficient MCF7 cells (shNIK1 and shNIK2) and MCF7 control cells (shLuc). All RT-qPCR were normalized to TBP. (n = 3, error bars are +/− s.d, p < 0.05). (G) IPA network of top gene networks from NIK-overexpressing cells. (H) IPA network of top gene networks from NIK-depleted cells. Note that ERK1/2 is a central node in both networks. Red color indicates induced genes and green color represents suppressed genes.

Mentions: To elucidate the possible mechanism by which NIK affects the stem cell phenotype, we performed whole-genome microarray expression analysis of NIK-overexpressing, NIK-deficient, and MCF7 control cells. As expected, the principal component analysis divided the samples into three groups; high NIK expression (NIK+), low NIK expression (NIK−) and the control cells (Fig. 8A). We found significant gene expression differences in 79 genes in MCF7 overexpressing-NIK cells and 53 differential expressed genes in MCF7 deficient NIK cells (Supplementary Table S2 and S3 and Fig. 8B). Supporting the role of NIK in CSCs regulation, we found that most of the top regulated genes were involved in stem cell or EMT processes (Fig. 8C,D).


NF-kappa Β -inducing kinase regulates stem cell phenotype in breast cancer
(A) Principal component analysis of gene expression profiles for high NIK-expressing MCF7 cells, low NIK-expressing MCF7 cells and MCF7 control cells. (B) Clustering diagram of samples and differentially expressed genes in MCF7 cells overexpressing NIK and NIK-deficient MCF7 cells. Log-Fold Changes of differentially expressed genes are depicted in a heat map, scaling from low (green) to high (red). Experiments were performed in three independent transiently-transfected MCF7 cell lines (C). Graph showing the fold change of stem cells-associated genes in high NIK-expressing cells and (D) low NIK-expressing cells. (E) RT-qPCR analysis of EGR1, TCN1, DUSP6, GDF15, CSPG4 and MAP2K6 in MCF7 overexpressing-NIK cells, MCF7 control cells, NIK-deficient MCF7 cells (shNIK1 and shNIK2) and MCF7 control cells (shLuc). All RT-qPCR were normalized to TBP. (n = 3, error bars are +/− s.d, p < 0.05). (G) IPA network of top gene networks from NIK-overexpressing cells. (H) IPA network of top gene networks from NIK-depleted cells. Note that ERK1/2 is a central node in both networks. Red color indicates induced genes and green color represents suppressed genes.
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f8: (A) Principal component analysis of gene expression profiles for high NIK-expressing MCF7 cells, low NIK-expressing MCF7 cells and MCF7 control cells. (B) Clustering diagram of samples and differentially expressed genes in MCF7 cells overexpressing NIK and NIK-deficient MCF7 cells. Log-Fold Changes of differentially expressed genes are depicted in a heat map, scaling from low (green) to high (red). Experiments were performed in three independent transiently-transfected MCF7 cell lines (C). Graph showing the fold change of stem cells-associated genes in high NIK-expressing cells and (D) low NIK-expressing cells. (E) RT-qPCR analysis of EGR1, TCN1, DUSP6, GDF15, CSPG4 and MAP2K6 in MCF7 overexpressing-NIK cells, MCF7 control cells, NIK-deficient MCF7 cells (shNIK1 and shNIK2) and MCF7 control cells (shLuc). All RT-qPCR were normalized to TBP. (n = 3, error bars are +/− s.d, p < 0.05). (G) IPA network of top gene networks from NIK-overexpressing cells. (H) IPA network of top gene networks from NIK-depleted cells. Note that ERK1/2 is a central node in both networks. Red color indicates induced genes and green color represents suppressed genes.
Mentions: To elucidate the possible mechanism by which NIK affects the stem cell phenotype, we performed whole-genome microarray expression analysis of NIK-overexpressing, NIK-deficient, and MCF7 control cells. As expected, the principal component analysis divided the samples into three groups; high NIK expression (NIK+), low NIK expression (NIK−) and the control cells (Fig. 8A). We found significant gene expression differences in 79 genes in MCF7 overexpressing-NIK cells and 53 differential expressed genes in MCF7 deficient NIK cells (Supplementary Table S2 and S3 and Fig. 8B). Supporting the role of NIK in CSCs regulation, we found that most of the top regulated genes were involved in stem cell or EMT processes (Fig. 8C,D).

View Article: PubMed Central - PubMed

ABSTRACT

Breast cancer stem cells (BCSCs) overexpress components of the Nuclear factor-kappa B (NF-&kappa;B) signaling cascade and consequently display high NF-&kappa;B activity levels. Breast cancer cell lines with high proportion of CSCs exhibit high NF-&kappa;B-inducing kinase (NIK) expression. The role of NIK in the phenotype of cancer stem cell regulation is poorly understood. Expression of NIK was analyzed by quantitative RT-PCR in BCSCs. NIK levels were manipulated through transfection of specific shRNAs or an expression vector. The effect of NIK in the cancer stem cell properties was assessed by mammosphere formation, mice xenografts and stem markers expression. BCSCs expressed higher levels of NIK and its inhibition through small hairpin (shRNA), reduced the expression of CSC markers and impaired clonogenicity and tumorigenesis. Genome-wide expression analyses suggested that NIK acts on ERK1/2 pathway to exert its activity. In addition, forced expression of NIK increased the BCSC population and enhanced breast cancer cell tumorigenicity. The in vivo relevance of these results is further supported by a tissue microarray of breast cancer samples in which we observed correlated expression of Aldehyde dehydrogenase (ALDH) and NIK protein. Our results support the essential involvement of NIK in BCSC phenotypic regulation via ERK1/2 and NF-&kappa;B.

No MeSH data available.


Related in: MedlinePlus