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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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Related in: MedlinePlus

Nuclear factor-kappa B-inducing kinase (NIK) role in the regulation of Cancer stem cell (CSC) population of MCF7 and MDA-MB-231.(A,B) Breast cancer stem cell (BCSC) populations derived from MCF7 and MDA-MB-_231 are enriched in NIK. RT-qPCR analysis demonstrated that NIK is mainly expressed in the BCSC population of MCF7 (A) (n = 3, error bars are +/− s.e.m) and MDAMB231 (B) (n = 2, error bars are +/− s.e.m) cell lines. To knockdown NIK, two short hairpin RNA (shRNA) were used in MCF7 (C) and MDA-MB231 (D) cell lines. (n = 3, error bars are +/− s.e.m, *P < 0.05). (E) The impact of NIK expression over CSC markers levels was evaluated after stable inhibition of NIK in two independent MCF7 replicates. Real Time PCR analysis shows that NIK depletion affects the expression of CSC markers (SOX2, OCT4, ALDH1A3, ALDH8A1). (n = 2, error bars are +/− s.e.m). (F) Effect of NIK inhibition in two independent MDA-MB-231 stable transfected cells lines with shNIK1 or shNIK2. RT-qPCR analysis of markers expression after stable NIK inhibition in MDA-MB-231 cells. All Real Time PCR were normalized to TBP. The level of CSC markers or NIK was designated to 1 for MCF7 or MDA-MB-231 control cells (shLuc). (n = 2, error bars are +/− s.e.m).
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f2: Nuclear factor-kappa B-inducing kinase (NIK) role in the regulation of Cancer stem cell (CSC) population of MCF7 and MDA-MB-231.(A,B) Breast cancer stem cell (BCSC) populations derived from MCF7 and MDA-MB-_231 are enriched in NIK. RT-qPCR analysis demonstrated that NIK is mainly expressed in the BCSC population of MCF7 (A) (n = 3, error bars are +/− s.e.m) and MDAMB231 (B) (n = 2, error bars are +/− s.e.m) cell lines. To knockdown NIK, two short hairpin RNA (shRNA) were used in MCF7 (C) and MDA-MB231 (D) cell lines. (n = 3, error bars are +/− s.e.m, *P < 0.05). (E) The impact of NIK expression over CSC markers levels was evaluated after stable inhibition of NIK in two independent MCF7 replicates. Real Time PCR analysis shows that NIK depletion affects the expression of CSC markers (SOX2, OCT4, ALDH1A3, ALDH8A1). (n = 2, error bars are +/− s.e.m). (F) Effect of NIK inhibition in two independent MDA-MB-231 stable transfected cells lines with shNIK1 or shNIK2. RT-qPCR analysis of markers expression after stable NIK inhibition in MDA-MB-231 cells. All Real Time PCR were normalized to TBP. The level of CSC markers or NIK was designated to 1 for MCF7 or MDA-MB-231 control cells (shLuc). (n = 2, error bars are +/− s.e.m).

Mentions: NIK (also known as MAP3K14), a NF-κB activator, is upregulated in basal breast cancer cell lines1920, which are enriched in CSCs22. For this reason, we examined the levels of this kinase in MDA-MB-231 (claudin low), SKBR3 (HER2+) and MCF7 (luminal) breast cancer cell lines and found that as expected, NIK is highly expressed in MDA-MB-231 cells and barely detectable in MCF7 cells (Supplementary Fig. S2). To determine if NIK expression in BCSCs was up-regulated, we examined its mRNA levels in a two cell lines. Figure 2A shows that MCF7 BCSCs expressed higher levels of NIK than the non-stem cell population (CD44−/CD24+). In addition, MDA-MB-231 BCSCs also exhibited higher levels of NIK when compared to CD44−/ESA− cells (Fig. 2B); furthermore NIK is highly expressed in MDA-MB-231 cells with ALDH activity (Supplementary Fig. S1B). These results prompted us to investigate the role of NIK in the stem phenotype of breast cancer.


NF-kappa Β -inducing kinase regulates stem cell phenotype in breast cancer
Nuclear factor-kappa B-inducing kinase (NIK) role in the regulation of Cancer stem cell (CSC) population of MCF7 and MDA-MB-231.(A,B) Breast cancer stem cell (BCSC) populations derived from MCF7 and MDA-MB-_231 are enriched in NIK. RT-qPCR analysis demonstrated that NIK is mainly expressed in the BCSC population of MCF7 (A) (n = 3, error bars are +/− s.e.m) and MDAMB231 (B) (n = 2, error bars are +/− s.e.m) cell lines. To knockdown NIK, two short hairpin RNA (shRNA) were used in MCF7 (C) and MDA-MB231 (D) cell lines. (n = 3, error bars are +/− s.e.m, *P < 0.05). (E) The impact of NIK expression over CSC markers levels was evaluated after stable inhibition of NIK in two independent MCF7 replicates. Real Time PCR analysis shows that NIK depletion affects the expression of CSC markers (SOX2, OCT4, ALDH1A3, ALDH8A1). (n = 2, error bars are +/− s.e.m). (F) Effect of NIK inhibition in two independent MDA-MB-231 stable transfected cells lines with shNIK1 or shNIK2. RT-qPCR analysis of markers expression after stable NIK inhibition in MDA-MB-231 cells. All Real Time PCR were normalized to TBP. The level of CSC markers or NIK was designated to 1 for MCF7 or MDA-MB-231 control cells (shLuc). (n = 2, error bars are +/− s.e.m).
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Related In: Results  -  Collection

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f2: Nuclear factor-kappa B-inducing kinase (NIK) role in the regulation of Cancer stem cell (CSC) population of MCF7 and MDA-MB-231.(A,B) Breast cancer stem cell (BCSC) populations derived from MCF7 and MDA-MB-_231 are enriched in NIK. RT-qPCR analysis demonstrated that NIK is mainly expressed in the BCSC population of MCF7 (A) (n = 3, error bars are +/− s.e.m) and MDAMB231 (B) (n = 2, error bars are +/− s.e.m) cell lines. To knockdown NIK, two short hairpin RNA (shRNA) were used in MCF7 (C) and MDA-MB231 (D) cell lines. (n = 3, error bars are +/− s.e.m, *P < 0.05). (E) The impact of NIK expression over CSC markers levels was evaluated after stable inhibition of NIK in two independent MCF7 replicates. Real Time PCR analysis shows that NIK depletion affects the expression of CSC markers (SOX2, OCT4, ALDH1A3, ALDH8A1). (n = 2, error bars are +/− s.e.m). (F) Effect of NIK inhibition in two independent MDA-MB-231 stable transfected cells lines with shNIK1 or shNIK2. RT-qPCR analysis of markers expression after stable NIK inhibition in MDA-MB-231 cells. All Real Time PCR were normalized to TBP. The level of CSC markers or NIK was designated to 1 for MCF7 or MDA-MB-231 control cells (shLuc). (n = 2, error bars are +/− s.e.m).
Mentions: NIK (also known as MAP3K14), a NF-κB activator, is upregulated in basal breast cancer cell lines1920, which are enriched in CSCs22. For this reason, we examined the levels of this kinase in MDA-MB-231 (claudin low), SKBR3 (HER2+) and MCF7 (luminal) breast cancer cell lines and found that as expected, NIK is highly expressed in MDA-MB-231 cells and barely detectable in MCF7 cells (Supplementary Fig. S2). To determine if NIK expression in BCSCs was up-regulated, we examined its mRNA levels in a two cell lines. Figure 2A shows that MCF7 BCSCs expressed higher levels of NIK than the non-stem cell population (CD44−/CD24+). In addition, MDA-MB-231 BCSCs also exhibited higher levels of NIK when compared to CD44−/ESA− cells (Fig. 2B); furthermore NIK is highly expressed in MDA-MB-231 cells with ALDH activity (Supplementary Fig. S1B). These results prompted us to investigate the role of NIK in the stem phenotype of breast cancer.

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