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.
Highlights
NIK, a MAP kinase kinase kinase (MAP3K14) protein, is essential for the activation of the non-canonical NF-κBpathway because it phosphorylates IκBKinase-α(IKKα)and participates in the processing of p10010
By disrupting NIK expression, we show that NIK inhibition affects the number of Breast Cancer Stem Cells (BCSCs) and concomitantly reduces the expression levels of Aldehyde Dehydrogrenase-1A1 (ALDH1A1), NANOG, SOX2 (SRY-BOX2), and Octamer-Binding Transcription Factor (OCT4)
We demonstrated that MCF7 cell line has a low percentage of BCSCs (CD44+/CD24−/low cells; 0.7–1.4%; Fig. 1A)
Summary
NIK, a MAP kinase kinase kinase (MAP3K14) protein, is essential for the activation of the non-canonical NF-κBpathway because it phosphorylates IκBKinase-α(IKKα)and participates in the processing of p10010. NIK is involved in processes such as cell differentiation, development, and embryogenesis; in the latter, NIK appears to play a role in pluripotent embryonic stem cell maintenance[12] These activities of NIK support a potential role in the regulation of stem cell behavior[12,13,14,15]. In this regard, mutant mice with defects in the non-canonical NF-κB pathway, including NIK, display abnormalities in mammary gland development[16,17,18]. NIK inhibition impaired the ability of cells to grow tumors in immunodeficient mice In support of these results, we observed that NIK overexpression increased the proportion of CD44+/CD24−/low cells and stem cell markers in MCF7 cells. Microarray data revealed that NIK regulated stem cell-related genes through the Extracellular Signal-Regulates Kinases (ERK) pathway
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