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Comprehensive Evaluation of Nuclear Factor-κΒ Expression Patterns in Non-Small Cell Lung Cancer.

Giopanou I, Lilis I, Papaleonidopoulos V, Marazioti A, Spella M, Vreka M, Papadaki H, Stathopoulos GT - PLoS ONE (2015)

Bottom Line: We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach.Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100.We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Achaia, Greece.

ABSTRACT
Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC.

No MeSH data available.


Related in: MedlinePlus

Association of NF-κB subunit expression with tumor-related inflammation and cellular proliferation in NSCLC.(A) Representative images of hematoxylin-stained samples showing different degrees of inflammatory infiltration of stroma areas. (B) NF-κB subunit expression scores of tumors with varying degrees of inflammatory infiltration. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, **, and ***: P > 0.05, P < 0.01, and P < 0.001 for indicated comparisons by Kruskal-Wallis tests followed by Dunn’s post-tests. (C) Representative images of PCNA-stained NSCLC subtype samples. (D) Nuclear co-localization of PCNA immunoreactivity with RelB (arrows), but not with RelA, was identified using dual immunostaining of samples of 10 patients (representative images shown).
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pone.0132527.g003: Association of NF-κB subunit expression with tumor-related inflammation and cellular proliferation in NSCLC.(A) Representative images of hematoxylin-stained samples showing different degrees of inflammatory infiltration of stroma areas. (B) NF-κB subunit expression scores of tumors with varying degrees of inflammatory infiltration. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, **, and ***: P > 0.05, P < 0.01, and P < 0.001 for indicated comparisons by Kruskal-Wallis tests followed by Dunn’s post-tests. (C) Representative images of PCNA-stained NSCLC subtype samples. (D) Nuclear co-localization of PCNA immunoreactivity with RelB (arrows), but not with RelA, was identified using dual immunostaining of samples of 10 patients (representative images shown).

Mentions: Since NF-κB is mechanistically implicated in the proliferation and inflammatory signalling of lung tumor cells in mouse models [21–24], we sought to determine the degree of cellular proliferation and inflammatory infiltration in our 77 patients with NSCLC. For this, serial sections were subjected to PCNA immunostaining as above, with simultaneous hematoxylin staining of isotype control sections, and the percentage of PCNA-positive cells in tumor areas was assessed as a measure of cellular proliferation rate. In addition, the percentage of tumor-infiltrating PCNA-negative inflammatory cells in stroma areas was determined for each patient (Fig 3A). These analyses identified that, although expression levels of RelA in tumor areas were quite low, they were increased in tumors with high levels of inflammatory infiltration (Fig 3B). Moreover, although the percentage of PCNA-positive cells was not different between the different histologic subtypes (Fig 3C), and was not associated with the expression of any NF-κB subunit (data not shown), dual immunofluorescence for RelA or RelB combined with PCNA revealed that PCNA+ proliferating cells predominantly expressed RelB in their nucleus (Fig 3D). These data indicated that tumor RelA expression was associated with inflammatory infiltration and that tumor RelB expression was linked with cellular proliferation within each tumor. Since RelB expression has been found to be regulated by both the canonical as well as the alternative NF-κB pathways, we next sought to examine a possible correlation between RelA expression in tumor or stroma compartments, and RelB expression in tumor cells. However, no statistically significant correlation was found when the data were assessed either in a parametric or a non-parametric fashion, suggesting that tumor RelA and RelB expression occur independently in NSCLC.


Comprehensive Evaluation of Nuclear Factor-κΒ Expression Patterns in Non-Small Cell Lung Cancer.

Giopanou I, Lilis I, Papaleonidopoulos V, Marazioti A, Spella M, Vreka M, Papadaki H, Stathopoulos GT - PLoS ONE (2015)

Association of NF-κB subunit expression with tumor-related inflammation and cellular proliferation in NSCLC.(A) Representative images of hematoxylin-stained samples showing different degrees of inflammatory infiltration of stroma areas. (B) NF-κB subunit expression scores of tumors with varying degrees of inflammatory infiltration. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, **, and ***: P > 0.05, P < 0.01, and P < 0.001 for indicated comparisons by Kruskal-Wallis tests followed by Dunn’s post-tests. (C) Representative images of PCNA-stained NSCLC subtype samples. (D) Nuclear co-localization of PCNA immunoreactivity with RelB (arrows), but not with RelA, was identified using dual immunostaining of samples of 10 patients (representative images shown).
© Copyright Policy
Related In: Results  -  Collection

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pone.0132527.g003: Association of NF-κB subunit expression with tumor-related inflammation and cellular proliferation in NSCLC.(A) Representative images of hematoxylin-stained samples showing different degrees of inflammatory infiltration of stroma areas. (B) NF-κB subunit expression scores of tumors with varying degrees of inflammatory infiltration. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns, **, and ***: P > 0.05, P < 0.01, and P < 0.001 for indicated comparisons by Kruskal-Wallis tests followed by Dunn’s post-tests. (C) Representative images of PCNA-stained NSCLC subtype samples. (D) Nuclear co-localization of PCNA immunoreactivity with RelB (arrows), but not with RelA, was identified using dual immunostaining of samples of 10 patients (representative images shown).
Mentions: Since NF-κB is mechanistically implicated in the proliferation and inflammatory signalling of lung tumor cells in mouse models [21–24], we sought to determine the degree of cellular proliferation and inflammatory infiltration in our 77 patients with NSCLC. For this, serial sections were subjected to PCNA immunostaining as above, with simultaneous hematoxylin staining of isotype control sections, and the percentage of PCNA-positive cells in tumor areas was assessed as a measure of cellular proliferation rate. In addition, the percentage of tumor-infiltrating PCNA-negative inflammatory cells in stroma areas was determined for each patient (Fig 3A). These analyses identified that, although expression levels of RelA in tumor areas were quite low, they were increased in tumors with high levels of inflammatory infiltration (Fig 3B). Moreover, although the percentage of PCNA-positive cells was not different between the different histologic subtypes (Fig 3C), and was not associated with the expression of any NF-κB subunit (data not shown), dual immunofluorescence for RelA or RelB combined with PCNA revealed that PCNA+ proliferating cells predominantly expressed RelB in their nucleus (Fig 3D). These data indicated that tumor RelA expression was associated with inflammatory infiltration and that tumor RelB expression was linked with cellular proliferation within each tumor. Since RelB expression has been found to be regulated by both the canonical as well as the alternative NF-κB pathways, we next sought to examine a possible correlation between RelA expression in tumor or stroma compartments, and RelB expression in tumor cells. However, no statistically significant correlation was found when the data were assessed either in a parametric or a non-parametric fashion, suggesting that tumor RelA and RelB expression occur independently in NSCLC.

Bottom Line: We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach.Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100.We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Achaia, Greece.

ABSTRACT
Nuclear factor (NF)-κB signalling is required for lung adenocarcinoma development in mice, and both of its subunits RelA and RelB were independently reported to be highly expressed in human non-small cell lung cancer (NSCLC). To comprehensively examine NF-κB expression in NSCLC, we analyzed serial sections of primary tumor samples from 77 well-documented patients (36 adenocarcinomas, 40 squamous cell carcinomas and 3 large cell carcinomas) for immunoreactivity of RelA, RelB, P50, and P52/P100. Tumor and intratumoral stroma areas were discriminated based on proliferating cell nuclear antigen immunoreactivity and inflammatory infiltration was assessed in intratumoral stroma areas. NF-κB immunoreactivity was quantified by intensity, extent, and nuclear localization and was cross-examined with tumor cell proliferation, inflammatory infiltration, and clinical-pathologic data. We found that the expression of the different NF-κB subunits was not concordant, warranting our integral approach. Overall, RelA, RelB, and P50 were expressed at higher levels compared with P52/P100. However, RelA and P50 were predominantly expressed in intratumoral stroma, but RelB in tumor cells. Importantly, tumor area RelA expression was correlated with the intensity of inflammatory infiltration, whereas RelB expression was identified in proliferating tumor cells. Using multiple logistic regression, we identified that tumor RelB expression was an independent predictor of lymph node metastasis, and tumor P50 was an independent predictor of TNM6 stage IIB or higher, whereas tumor RelA was an independent predictor of inflammatory infiltration. We conclude that pathologic studies of NF-κB expression in cancer should include multiple pathway components. Utilizing such an approach, we identified intriguing associations between distinct NF-κB subunits and clinical and pathologic features of NSCLC.

No MeSH data available.


Related in: MedlinePlus