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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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Immunohistochemistry analysis of NIK and Aldehyde dehydrogenase (ALDH) expression in breast carcinomas (n = 191).(A) Percentage of Nuclear factor-kappa B-inducing kinase (NIK)-positive breast tissues. (B) Percentage of ALDH positive breast tissues. (C) Venn diagram showing breast cancer tissues expressing both NIK and ALDH protein. (D–H) Immunohistochemistry of NIK and Aldehyde dehydrogenase (ALDH) expression. NIK-positive cells are brown based on DAB chromogen, and ALDH-positive cells are pink based on Fast Red chromogen. Nuclei are stained with hematoxylin (blue). The second column represents an enlargement of the area denoted by box in the adjacent left panel showing fine cellular details of immunohistochemistry. (D) Graph showing percentage of tissues having cellular colocalization in Breast Cancer samples expressing both NIK and ALDH. (E) Representation of breast tumors (n = 93) expressing ALDH and NIK in the same cells. (F) Immunohistochemistry of breast tumors showing NIK and ALDH expression in different cells (n = 10).
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f7: Immunohistochemistry analysis of NIK and Aldehyde dehydrogenase (ALDH) expression in breast carcinomas (n = 191).(A) Percentage of Nuclear factor-kappa B-inducing kinase (NIK)-positive breast tissues. (B) Percentage of ALDH positive breast tissues. (C) Venn diagram showing breast cancer tissues expressing both NIK and ALDH protein. (D–H) Immunohistochemistry of NIK and Aldehyde dehydrogenase (ALDH) expression. NIK-positive cells are brown based on DAB chromogen, and ALDH-positive cells are pink based on Fast Red chromogen. Nuclei are stained with hematoxylin (blue). The second column represents an enlargement of the area denoted by box in the adjacent left panel showing fine cellular details of immunohistochemistry. (D) Graph showing percentage of tissues having cellular colocalization in Breast Cancer samples expressing both NIK and ALDH. (E) Representation of breast tumors (n = 93) expressing ALDH and NIK in the same cells. (F) Immunohistochemistry of breast tumors showing NIK and ALDH expression in different cells (n = 10).

Mentions: Immunohistochemistry analysis revealed that NIK was expressed in 79.5% (152) breast cancer tissues (Fig. 7A). To support our previous result showing that NIK and ALDH expression are correlated, we analyzed the co-localization of both proteins in these samples. ALDH was expressed in 56% of the studied cases (Fig. 7B), of which 97% also expressed NIK (Fig. 7C). Surprisingly, 89.4% of those tumors presented a cellular co-localization of both proteins as shown in Fig. 7D. Figure 7E represents tumors that express NIK and ALDH in different cells. Figure 7F–H represents tumors lacking either NIK or ALDH.


NF-kappa Β -inducing kinase regulates stem cell phenotype in breast cancer
Immunohistochemistry analysis of NIK and Aldehyde dehydrogenase (ALDH) expression in breast carcinomas (n = 191).(A) Percentage of Nuclear factor-kappa B-inducing kinase (NIK)-positive breast tissues. (B) Percentage of ALDH positive breast tissues. (C) Venn diagram showing breast cancer tissues expressing both NIK and ALDH protein. (D–H) Immunohistochemistry of NIK and Aldehyde dehydrogenase (ALDH) expression. NIK-positive cells are brown based on DAB chromogen, and ALDH-positive cells are pink based on Fast Red chromogen. Nuclei are stained with hematoxylin (blue). The second column represents an enlargement of the area denoted by box in the adjacent left panel showing fine cellular details of immunohistochemistry. (D) Graph showing percentage of tissues having cellular colocalization in Breast Cancer samples expressing both NIK and ALDH. (E) Representation of breast tumors (n = 93) expressing ALDH and NIK in the same cells. (F) Immunohistochemistry of breast tumors showing NIK and ALDH expression in different cells (n = 10).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120353&req=5

f7: Immunohistochemistry analysis of NIK and Aldehyde dehydrogenase (ALDH) expression in breast carcinomas (n = 191).(A) Percentage of Nuclear factor-kappa B-inducing kinase (NIK)-positive breast tissues. (B) Percentage of ALDH positive breast tissues. (C) Venn diagram showing breast cancer tissues expressing both NIK and ALDH protein. (D–H) Immunohistochemistry of NIK and Aldehyde dehydrogenase (ALDH) expression. NIK-positive cells are brown based on DAB chromogen, and ALDH-positive cells are pink based on Fast Red chromogen. Nuclei are stained with hematoxylin (blue). The second column represents an enlargement of the area denoted by box in the adjacent left panel showing fine cellular details of immunohistochemistry. (D) Graph showing percentage of tissues having cellular colocalization in Breast Cancer samples expressing both NIK and ALDH. (E) Representation of breast tumors (n = 93) expressing ALDH and NIK in the same cells. (F) Immunohistochemistry of breast tumors showing NIK and ALDH expression in different cells (n = 10).
Mentions: Immunohistochemistry analysis revealed that NIK was expressed in 79.5% (152) breast cancer tissues (Fig. 7A). To support our previous result showing that NIK and ALDH expression are correlated, we analyzed the co-localization of both proteins in these samples. ALDH was expressed in 56% of the studied cases (Fig. 7B), of which 97% also expressed NIK (Fig. 7C). Surprisingly, 89.4% of those tumors presented a cellular co-localization of both proteins as shown in Fig. 7D. Figure 7E represents tumors that express NIK and ALDH in different cells. Figure 7F–H represents tumors lacking either NIK or ALDH.

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.

No MeSH data available.


Related in: MedlinePlus