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. 2021 May 19:11:638856.
doi: 10.3389/fonc.2021.638856. eCollection 2021.

The Ferroxidase Hephaestin in Lung Cancer: Pathological Significance and Prognostic Value

Paola Zacchi  1 , Beatrice Belmonte  2 , Alessandro Mangogna  3 , Gaia Morello  2 , Letizia Scola  4 , Anna Martorana  2 , Violetta Borelli  1
Affiliations

The Ferroxidase Hephaestin in Lung Cancer: Pathological Significance and Prognostic Value

Paola Zacchi et al. Front Oncol. .

Abstract

Hephaestin (HEPH) belongs to a group of exocytoplasmic ferroxidases which contribute to cellular iron homeostasis by favouring its export. Down-regulation of HEPH expression, possibly by stimulating cell proliferation due to an increase in iron availability, has shown to correlate with poor survival in breast cancer. The lung is particularly sensitive to iron-induced oxidative stress, given the high oxygen tension present, however, HEPH distribution in lung cancer and its influence on prognosis have not been investigated yet. In this study we explored the prognostic value of HEPH and its expression pattern in the most prevalent histotypes of lung cancers, namely lung adenocarcinoma and lung squamous cell carcinoma. In silico analyses, based on UALCAN, Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan-Meier plotter bioinformatics, revealed a significant correlation between higher levels of HEPH expression and favorable prognosis, in both cancer histotypes. Moreover, TIMER web platform showed a statistically significant association between HEPH expression and cell elements belonging to the tumor microenvironment identified as endothelial cells and a subpopulation of cancer-associated fibroblasts, further confirmed by double immunohistochemical labeling with cell type specific markers. Taken together, these data shed a light on the complex mechanisms of local iron handling lung cancer can exploit to support tumorigenesis.

Keywords: bioinformatics; hephaestin; immunohistochemistry; iron; lung cancer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Pathological significance of HEPH expression in different types of human cancer, and in-depth evaluation in LUAD and LUSC. (A) Human HEPH expression levels in different tumor types from TCGA database were determined by TIMER (*P < 0.05, **P < 0.01, ***P < 0.001). (B) HEPH mRNA expression comparisons between normal (blue) and tumor tissues (red) obtained from the GEPIA web tool. (C) HEPH protein expression comparison between normal and tumor tissues obtained from the UALCAN web tool (Wilcoxon test). P-value <0.05 was used to assess differences. (D) Survival analyses of HEPH by Kaplan–Meier estimator with log-rank test obtained from the Kaplan–Meier plotter web tool. Survival differences are compared between patients with high (red) and low (black) HEPH expression (grouped according to Auto select best cut-off). H, high expression; L, low expression.
Figure 2
Figure 2
Correlation of HEPH expression with infiltration level of non-immune cells in LUAD and LUSC. HEPH expression is significantly negatively correlated to tumor purity and has significant positive correlations with the infiltrating levels of cancer-associated fibroblasts and endothelial cells.
Figure 3
Figure 3
HEPH expression positively correlated with markers of cancer-associated fibroblasts (A) and endothelial cells (B) in both LUAD and LUSC. Scatterplots of correlations between HEPH and gene markers include ACTA2, FAP, PDGFRA, PDGFRB for cancer-associated fibroblasts and PECAM1 (CD31) and vWF for endothelial cells.
Figure 4
Figure 4
Comparisons of the mRNA expression levels of cancer-associated fibfroblasts and endothelial cells markers between normal (grey) and tumor tissues (red) (A). Overall survival curve of each cancer-associated fibroblasts and endothelial marker shown to correlate with HEPH expression and produced by Kaplan–Meier website resource (B). OS differences are compared between patients with high and low HEPH expression (grouped according to Auto select best cut-off). H, high expression; L, low expression.
Figure 5
Figure 5
HEPH distribution in control non-tumor lung and in LUAD and LUSC specimens. Representative microphotographs relative to HEPH distribution in non-tumor lung (A–D). In panel (B) an HEPH expressing type II pneumocyte is indicated by arrow-head while a black arrow points to smooth muscle fibers and bronchiolar epithelium. In panel (D) endothelial cells are indicated by an arrow. HEPH distribution by cancer cells in the context of the two histotypes. Panels (E) (LUAD) and (G) (LUSC) show the tumoral areas in which HEPH is poorly or not expressed. Panels (F) (LUAD) and (H) (LUSC) correspond to cancer nests expressing HEPH. Polymer detection system with AEC (red) chromogen; scale bars, 50 µm.
Figure 6
Figure 6
HEPH expression by endothelial and stromal cells in LUAD and LUSC specimens. Endothelial cells identified morphologically in single HEPH immunostaining (panels (A, B) for LUAD and (C, D) for LUSC), and by way of the strong expression of PECAM1 (CD31) upon double-labeling (panels (I, J) for LUAD and (L, M) for LUSC). Black arrows indicate HEPH/PECAM1 colocalization. Fibroblasts identified based on their spindle-shaped morphology in single HEPH immunostaining [panels (E, F) for LUAD and (G, H) for LUSC], and by the expression of PDGFR-β upon double-labeling. Back arrows indicate HEPH/PDGFR-β colocalization [panels (M, N) for LUAD and (O, P) for LUSC]. Panels (J, M, N, P) represent higher magnifications of the corresponding dashed area indicated on the corresponding upper panel. Polymer detection system with AEC (red) chromogen for HEPH and DAB (3,3′-Diaminobenzidine) chromogen for PECAM1; scale bars, 50 µm.
Figure 7
Figure 7
HEPH distribution in tumor-associated macrophages. Representative microphotographs relative to HEPH expression in monocytes/macrophages recognized by CD14 immunoreactivity in LUAD (A, B) and LUSC (C, D). Black arrows indicate HEPH/CD14 colocalization. Polymer detection system with AEC (red) chromogen for HEPH and DAB (3,3′ -Diaminobenzidine) chromogen for CD14; scale bars, 50 µm.
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