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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

NF-κB subunit expression patterns in tumor versus intratumoral stroma areas.(A) Representative images. (B, D) Scoring of NF-κB subunit expression levels in tumor (B) and stroma (D) areas. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns and ***: P > 0.05 and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. (C, E) Co-expression matrixes of categorical NF-κB subunit expression levels in tumor (C) and stroma (E) areas. For this, NF-κB scores from (B) and (D) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 and P: probability values by χ2 tests followed by Fisher’s exact tests. (F) Co-expression matrixes of tumor versus stroma NF-κB subunit expression. ns: P > 0.05 and P: probability values by χ2 tests followed by Fisher’s exact tests. (G) Correlation of tumor and stroma P100/P52 expression scores. Shown are data points, linear regression line with 95% confidence interval, squared Spearman’s correlation coefficient, and probability value.
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pone.0132527.g002: NF-κB subunit expression patterns in tumor versus intratumoral stroma areas.(A) Representative images. (B, D) Scoring of NF-κB subunit expression levels in tumor (B) and stroma (D) areas. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns and ***: P > 0.05 and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. (C, E) Co-expression matrixes of categorical NF-κB subunit expression levels in tumor (C) and stroma (E) areas. For this, NF-κB scores from (B) and (D) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 and P: probability values by χ2 tests followed by Fisher’s exact tests. (F) Co-expression matrixes of tumor versus stroma NF-κB subunit expression. ns: P > 0.05 and P: probability values by χ2 tests followed by Fisher’s exact tests. (G) Correlation of tumor and stroma P100/P52 expression scores. Shown are data points, linear regression line with 95% confidence interval, squared Spearman’s correlation coefficient, and probability value.

Mentions: To study this in more detail, all samples were stained for PCNA, which clearly identified tumor areas (high abundance of PCNA+ cells) from the intratumoral stroma compartment (very low abundance of PCNA+ cells), and NF-κB subunit scoring was repeated separately for these two areas in serial sections of all patients (Fig 2A). These analyses revealed that RelB was the subunit expressed the strongest in areas of tumor cells displaying a both nuclear and cytoplasmic expression pattern. On the other hand, the remaining subunits studied showed only moderate cytoplasmic expression in tumor areas (Fig 2A and 2B). When tumor compartment NF-κB subunit scores were subdivided into low (0–4), intermediate (5–6), or high (7–18) and compared within each tumor, RelB and P50 showed significant discordance, with tumors with strong RelB immunoreactivity exhibiting low P50 scores (Fig 2C). Surprisingly, evaluation exclusively of the intratumoral stroma yielded different results: here, RelA and P50 subunits were most predominantly expressed in the nuclei of stromal cells. In stark contrast, RelB and P100/P52 showed only weak cytoplasmic immunoreactivity (Fig 2A and 2D). When stroma NF-κB subunit scores were subdivided into low (0–4), intermediate (5–6), or high (7–18) and compared within each tumor, RelB and P100/P52 showed significant concordance, with 49/77 tumors displaying simultaneously low scores for both subunits, likely reflecting the low expression levels of both proteins (Fig 2E). We finally examined the relationships between numerical and categorical tumor and stroma scores for each NF-κB subunit, finding that only P100/P52 expression in tumor and the related stroma were concordant and correlated (Fig 2F and 2G).


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)

NF-κB subunit expression patterns in tumor versus intratumoral stroma areas.(A) Representative images. (B, D) Scoring of NF-κB subunit expression levels in tumor (B) and stroma (D) areas. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns and ***: P > 0.05 and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. (C, E) Co-expression matrixes of categorical NF-κB subunit expression levels in tumor (C) and stroma (E) areas. For this, NF-κB scores from (B) and (D) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 and P: probability values by χ2 tests followed by Fisher’s exact tests. (F) Co-expression matrixes of tumor versus stroma NF-κB subunit expression. ns: P > 0.05 and P: probability values by χ2 tests followed by Fisher’s exact tests. (G) Correlation of tumor and stroma P100/P52 expression scores. Shown are data points, linear regression line with 95% confidence interval, squared Spearman’s correlation coefficient, and probability value.
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pone.0132527.g002: NF-κB subunit expression patterns in tumor versus intratumoral stroma areas.(A) Representative images. (B, D) Scoring of NF-κB subunit expression levels in tumor (B) and stroma (D) areas. Data presented as median with boxes indicating interquartile range and whiskers indicating 95% percentiles. ns and ***: P > 0.05 and P < 0.001 for indicated comparisons by Friedman’s test followed by Dunn’s post-tests. (C, E) Co-expression matrixes of categorical NF-κB subunit expression levels in tumor (C) and stroma (E) areas. For this, NF-κB scores from (B) and (D) were categorized into low (0–4), intermediate (5–6), and high (7–18). ns: P > 0.05 and P: probability values by χ2 tests followed by Fisher’s exact tests. (F) Co-expression matrixes of tumor versus stroma NF-κB subunit expression. ns: P > 0.05 and P: probability values by χ2 tests followed by Fisher’s exact tests. (G) Correlation of tumor and stroma P100/P52 expression scores. Shown are data points, linear regression line with 95% confidence interval, squared Spearman’s correlation coefficient, and probability value.
Mentions: To study this in more detail, all samples were stained for PCNA, which clearly identified tumor areas (high abundance of PCNA+ cells) from the intratumoral stroma compartment (very low abundance of PCNA+ cells), and NF-κB subunit scoring was repeated separately for these two areas in serial sections of all patients (Fig 2A). These analyses revealed that RelB was the subunit expressed the strongest in areas of tumor cells displaying a both nuclear and cytoplasmic expression pattern. On the other hand, the remaining subunits studied showed only moderate cytoplasmic expression in tumor areas (Fig 2A and 2B). When tumor compartment NF-κB subunit scores were subdivided into low (0–4), intermediate (5–6), or high (7–18) and compared within each tumor, RelB and P50 showed significant discordance, with tumors with strong RelB immunoreactivity exhibiting low P50 scores (Fig 2C). Surprisingly, evaluation exclusively of the intratumoral stroma yielded different results: here, RelA and P50 subunits were most predominantly expressed in the nuclei of stromal cells. In stark contrast, RelB and P100/P52 showed only weak cytoplasmic immunoreactivity (Fig 2A and 2D). When stroma NF-κB subunit scores were subdivided into low (0–4), intermediate (5–6), or high (7–18) and compared within each tumor, RelB and P100/P52 showed significant concordance, with 49/77 tumors displaying simultaneously low scores for both subunits, likely reflecting the low expression levels of both proteins (Fig 2E). We finally examined the relationships between numerical and categorical tumor and stroma scores for each NF-κB subunit, finding that only P100/P52 expression in tumor and the related stroma were concordant and correlated (Fig 2F and 2G).

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