Metallothioneins and Megalin Expression Profiling in Premalignant and Malignant Oral Squamous Epithelial Lesions

Abstract

This study aimed to assess the relationship and possible interactions between metallothioneins (MTs) and megalin (LRP-2) in different grades of oral squamous cell carcinoma (OSCC) and premalignant lesions of the oral mucosa (oral leukoplakia and oral lichen planus). The study included archived samples of 114 patients and control subjects. Protein expression was examined by immunohistochemistry and immunofluorescence, and staining quantification was performed by ImageJ software. Protein interaction in cancer tissue was tested and visualized by proximity ligation assay. Mann-Whitney and Kruskal-Wallis tests were used to determine the significance of differences between each group, whereas Pearson correlation coefficient was performed to test correlation. Expression of both proteins differed significantly between each group showing the same pattern of gradual increasing from oral lichen planus to poorly differentiated OSCC. Moreover, MTs and megalin were found to co-express and interact in cancer tissue, and their expression positively correlated within the overall study group. Findings of prominent nuclear and chromosomal megalin expression suggest that it undergoes regulated intramembrane proteolysis upon MTs binding, indicating its ability to directly affect gene expression and cellular division in cancer tissue. The data obtained point to the onco-driving potential of MTs-megalin interaction.

Keywords: interaction; lichen planus; megalin; metallothioneins; oral leukoplakia; oral squamous cell carcinoma; regulated intramembrane proteolysis.

Figure 1

Metallothionein I/II expression in premalignant and malignant oral squamous epithelial lesions. Representative photomicrographs show immunohistochemical staining with anti-MT I/II antibody on paraffin-embedded sections of tissue samples obtained from subjects with healthy oral mucosa (A), patients with oral lichen planus (B), oral leukoplakia (C,D), grade I OSCC (E,F), grade II OSCC (G–I), mixed-grade (I/II) OSCC (J), and grade III OSCC (K–N). Arrows on I indicate pyknotic nuclei and apoptotic bodies in areas lacking MT I/II immunopositivity. Asterisks on J mark grade I (*) and grade II (**) cancer tissue in mixed-grade tumor. Arrows on M and N point to MT I/II immunopositive cancer cells infiltrating adjacent non-immunopositive healthy tissue at tumor edges. Magnifications: (E,G,J,K) × 100; (A–C,F,H,L,M,N) × 400; (D,I) × 1000.

Figure 2

Quantified metallothionein I/II staining intensities in premalignant and malignant oral squamous epithelial lesions. The measurements were made by ImageJ software and data are expressed in arbitrary units (AU) as the median of average gray values with range. HOM, healthy oral mucosa; OLP, oral lichen planus; OSCC, oral squamous cell carcinoma. * p < 0.000001 when each group is compared to the other.

Figure 3

Megalin expression in premalignant and malignant oral squamous epithelial lesions. Representative photomicrographs show immunohistochemical staining with the anti-megalin antibody on paraffin-embedded sections of tissue samples obtained from subjects with healthy oral mucosa (A), patients with oral lichen planus (B), oral leukoplakia (C), grade I OSCC (D–F), grade II OSCC (G–I), and grade III OSCC (J–L). Arrows on I and L indicate chromosomal megalin immunopositivity in mitotic cells. Magnifications: (D,G,J) × 100; (A–C,E,F,H,K) × 400; (I,L) × 1000.

Figure 4

Quantified megalin staining intensities in premalignant and malignant oral squamous epithelial lesions. The measurements were made by ImageJ software and data are expressed in arbitrary units (AU) as the median of average gray values with range. HOM, healthy oral mucosa; OLP, oral lichen planus; OSCC, oral squamous cell carcinoma. * p < 0.000001 when each group is compared to the other.

Figure 5

Co-expression and interaction of metallothionein I/II and megalin in oral squamous cell carcinoma. (A) Representative photomicrographs of double immunofluorescence staining with anti-MT I/II (red staining) and anti-megalin (green staining) antibodies on paraffin-embedded sections of the OSCC tissue. Blue marks DAPI staining of nuclei. Arrows indicate sites of membrane colocalization. Magnification: × 1000. (B) Representative photomicrographs obtained by proximity ligation assay (PLA) on paraffin-embedded sections of the OSCC tissue using anti-MT I/II and anti-megalin antibodies. Red fluorescent signals represent interaction sites. Blue marks DAPI staining of nuclei. Magnification: × 1000.

Figure 6

Correlation of metallothionein I/II and megalin staining intensities. Pearson correlation analysis included data obtained from the overall study group. Correlation coefficient (r) = 0.8 (p < 0.001).

Figure 7

Impact of smoking habit on metallothionein I/II (A) and megalin (B) expression. The measurements were made by ImageJ software and data are expressed in arbitrary units (AU) as the median of average gray values with range. HOM, healthy oral mucosa; OLP, oral lichen planus; OSCC, oral squamous cell carcinoma.