Expression profiles of metallothionein-I/II and megalin/LRP-2 in uterine cervical squamous lesions

Abstract

Metallothioneins (MTs) are phylogenetically old cysteine-rich proteins, which are implicated in a variety of physiological and pathological processes. Their growth-regulating, anti-apoptotic and anti-inflammatory functions have been attributed not only to intracellular free radical scavenging and to zinc and copper regulation but also to the ability of secreted MT to bind on surface lipoprotein receptor-megalin/LRP2, which enables the endocytosis of MT-I/II and a wide range of other functionally distinct ligands. In the present study, we analysed the expression pattern of both proteins in 55 cases of premalignant transformation of cervical squamous cells, i.e. in low- and high-grade squamous intraepithelial lesion (LSIL and HSIL). The data showed that in LSIL (cervical intraepithelial neoplasia CIN1; N = 25) MTs were present only in basal and parabasal cells and that megalin was only weakly expressed. In HSIL (CIN2; N = 15 and CIN 3/carcinoma in situ; N = 15), however, overexpression and co-localization of MT with megalin were found in the entire hyperplastic epithelium. Moreover, megalin immunoreactivity appeared on the glandular epithelium and vascular endothelium, as well as on lymphatic cells in stroma. Besides, multiple megalin-positive cells expressed phosphorylated Akt1, implying that MT- and/or megalin-dependent prosurvival signal transduction pathways might contribute to the development of severe cervical dysplasia. The data emphasize the diagnostic power of combined MT/megalin analysis in pre-cancer screening.

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

“…Despite great attention is recently drawn by the role of extracellular MTs, modes of their release and mechanisms mediating their effects are poorly understood and mostly limited to the CNS pathology [ 23 , 24 ]. The results obtained from this study comply with our recent findings on simultaneous gradual induction of MTs and megalin during the premalignant transformation of squamous epithelia of the uterine cervix [ 43 ], but, to the best of our knowledge, here we for the first time showed induction of megalin and its interaction with MTs in malignant carcinomatous tissue of epithelial origin. So far, only a few studies have addressed the attention on the possible role of megalin in tumorigenesis.…”

“…As described before, MTs binding to megalin may result with receptor-ligand complex internalization, thereby supplying cells with additional MTs [ 25 , 26 , 27 , 28 , 29 ], as well as with protein kinase B (AKT-1) activation [ 25 , 26 , 27 , 28 , 29 ], which provide mitogenic signals to the cells being at the same time protected from the apoptotic clearance due to the high MT content. Such a scenario is supported by the studies showing the positive correlation between phosphorylated AKT and MTs expression in OSCC tissue [ 52 ] and co-expression of phosphorylated AKT and megalin in squamous epithelia of premalignant uterine cervical lesions [ 43 ]. Furthermore, consistent findings of cytoplasmic megalin immunopositivity in our samples ( Figure 3 ) point to rapid endocytic trafficking of megalin, which has previously been described as a fast-recycling and slow-degrading receptor [ 65 ].…”

“…Before the staining procedure, antigen demasking was applied by heating tissue sections in 10-mM and pH 6.0 sodium citrate buffer. Staining was performed using highly sensitive and specific DAKO EnVision+System, Peroxidase (DAB) kit (DAKO Cytomation, Santa Clara, CA, USA) as previously described [ 43 ]. Briefly, after blockage of non-specific binding and endogenous peroxidase activity with commercial blocking solution (DAKO Cytomation, Santa Clara, CA, USA), slides were incubated with mouse monoclonal anti-MT I + II antibody (clone E9; Dako Cytomation, Santa Clara, CA, USA; diluted 1:100 with 1% BSA in PBS) or rabbit polyclonal anti-megalin antibody (H-245, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:200 with 1% BSA in PBS) for 12 h at 4 °C in a humid chamber.…”

“…However, megalin expression in cancer tissue and premalignant lesions remains understudied, and varying expression of megalin might be a clue to the inconsistent results on MTs relation to cancer aggressiveness, obtained when MTs expression was considered separately [ 34 ]. Aiming to expand still limited data on this topic and to complement our recent findings regarding uterine cervical squamous lesions [ 43 ], in the present study we analyzed, compared, and correlated expression profiles of MT I/II and megalin in different histological grades of oral squamous cell carcinoma (OSCC), as well as in oral lichen planus (OLP) and oral leukoplakia (OL) as the most common and well histologically defined oral potentially malignant disorders (OPMD) progressing in some cases to OSCC [ 44 , 45 ]. Assuming the possible interactions, we also assayed MTs/megalin co-expression and their ligation in carcinomatous tissue.…”

Megalin Expression in Primary Oral Squamous Cell Carcinoma Is Associated With the Presence of Lymph Node Metastases, Vascular Invasion and Lower Overall Survival

“…Despite megalin may, owing to its receptor versatility and ability to trigger gene-modulating signaling pathways [9], promote oncogenesis and tumor progression, research on its role in cancer pathobiology has been somewhat neglected so far. Only several studies have linked megalin with malignant transformation, and literature data offering possible mechanistic insights almost completely lacking [15,16,[19][20][21]. Pedersen et al showed acquisition of megalin expression in brain non-Hodgkinlymphoma [20], while Schuetz and colleagues found that clear cell renal cell carcinoma overexpressed megalin [21].…”