Overexpression of ADAM10 in oral squamous cell carcinoma with metastases

Introduction

Oral squamous cell carcinoma (OSCC) is the most common type of malignant tumour of the oral and maxillofacial region. Despite advances in diagnostics and treatment, OSCC is associated with a poor prognosis because of its high recurrence rate, frequent invasion, and metastasis [1, 2]. Oral leukoplakia (OLK) with features of dysplasia is a premalignant lesion of oral mucosa. Approximately 3-17% of OLKs transform into oral squamous cell carcinoma, and the risk of malignant transformation is usually proportional to the severity of dysplasia [3]. Therefore, investigation of the mechanism of development and progression is critical to treatment of OSCC.
The ADAMs (a disintegrin and metalloproteinase) are a zinc-dependent family of proteins that contain: an integrin-binding domain, an N-terminal signal sequence followed by a prodomain, a metalloproteinase domain, a disintegrin domain with cysteine-rich region, a transmembrane, and cytoplasmic tail [4, 5]. In the mammalian genome, approximately 40 ADAMs were identified and described [4]. Some ADAMs can be expressed in somatic cells and some are present on reproductive system cells (testis) suggesting tissue-specific function [4]. The importance of ADAMs is underscored by the fact that some of the most vital ADAM genes (ADAM10 and ADAM17) can be found in primitive chordates, Drosophila melanogaster, and in yeast, Schizosaccharomyces pombe [6, 7].
ADAMs can participate in both proteolytic cleavage and cellular adhesion of various cell surface molecules. Via the proteolytic cleavage, ADAMs are involved in regulating the activity of multiple membrane-bound proteins (receptors, cytokines, adhesion molecules, growth factors) [4, 5]. Therefore, ADAMs are important contributors to physiological and pathophysiological processes, and may be potential therapeutic targets in various diseases including cancer [4, 8].
Literature data shows that ADAM10, ADAM12, and ADAM17 may play a pivotal role in the pathogenesis and progression of cancers, including proliferation, migration, and invasion [5, 9, 10]. ADAM10 has potential implications for regulation of tumour growth and metastasis as well as for tumour angiogenesis through the rapid modulation of cell signalling pathways and cell adhesion [11]. Literature data demonstrates that ADAM10 contributes to cell proliferation by increasing the transcriptional activity of -catenin and modulating its nuclear translocation [12]. Overexpression of ADAM10 has been described in a various types of cancer, including melanoma, hepatocellular and pancreatic carcinoma, lung, gastric, bladder, and breast cancer [13, 14, 15, 16, 17, 18, 19]. It has been demonstrated that downregulation of ADAM10 in HepG2 cell line significantly suppresses proliferation, migration, and invasion, and decreases chemotherapy drug resistance in vitro and tumour growth in vivo [20, 21].
Although the immunoexpression of ADAM10 in various types of tumours has been extensively characterised, its precise role in oral carcinogenesis and association with microvessel density are not clear. Therefore, the aim of our study was to evaluate the immunoexpression of ADAM10 and microvessel density in oral leukoplakia (OLK with low-grade dysplasia – OLK-LG, OLK with high-grade dysplasia – OLK-HG), and oral squamous cell carcinomas with metastases (OSCC M(+)), and without metastases (OSCC M(–)). Another purpose was to find a possible association between immunoexpression of ADAM10 and the presence of the metastases and microvessel density.

Material and methods

Patients

The university review board approved the methods of the present retrospective study. The authors complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. The study was conducted under the assumption that research findings would be kept anonymous.
Eighty cases of OSCC, 24 cases of OLK, and 19 controls (normal mucosa) were sourced from archival tissue blocks of the Department of Pathomorphology, Medical University of Lodz, Poland. Normal oral mucosa from the floor of the mouth (non-cancer affected patients), originated from the Department of Plastic and Reconstructive Surgery, Medical University of Lodz. All tissue sections taken from postoperative material were routinely fixed in formalin, processed, embedded in paraffin, and stained with haematoxylin and eosin. Histopathological diagnoses were established according to the current standards [22]. The main criteria used for the selection of cases was an anatomical placement of lesions (the floor of the mouth for OSCC and gingiva for OLK). The histopathological grade of cases of OLK was moderate (15 cases with low-grade dysplasia – OLK-LG), to severe (9 cases with high-grade dysplasia – OLK-HG). All cases of OSCC were divided into two groups: OSCC M(–) (without metastases, n = 38) and OSCC M(+) (with metastases to regional lymph nodes or/and with distant metastases, n = 42). Cases of OSCC were graded according to the WHO classification (for OSCC M(–): G1 n = 3, G2 n = 34, G3 n = 1, and for OSCC M(+): G1 n = 0, G2 n = 36, G3 n = 6). The age range for the OSCC M(–) group was from 28 to 75 years (the mean ±SD = 59.36 ±10.69), for the OSCC M(+) group it was from 40 to 84 years (the mean ±SD = 59.79 ±11.56), for OLK it was from 21 to 84 years (mean ±SD = 50.02 ±11.14), and for controls it was from 15 to 74 years (mean ± SD = 45.25 ±19.02).

Immunohistochemistry

Immunohistochemical staining was carried out according to a standard method. Tissue sections 3 µm in size were deparaffinised in xylene and rehydrated through a graded alcohol series. Heating in a microwave oven in a solution of TRS (Target Retrieval Solution, pH 9.0, Dako) for 30 minutes was carried out for antigen retrieval. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide in methanol for 30 minutes. The sections were washed with TBS and incubated with monoclonal mouse primary antibodies against ADAM10 (Thermo Scientific, Rockford, USA, PA 5-28161, dilution 1 : 300) and CD31 (Dako, Glostrup, Denmark, clone JC70A, dilution 1:50). After washing, an adequate EnVision-HRP detection system (Dako, Carpinteria, CA, USA) was used. 3,3’-diaminobenzidine was used as the chromogen. After counterstaining with Mayer’s haematoxylin, the slides were washed, dehydrated, cleared in xylene, and coverslipped. The negative controls involved the same procedure, with the primary antibodies replaced by antibody diluent.
In each specimen distribution and cytoplasmic staining intensity of ADAM 10 in epithelial cells were recorded semiquantitatively by two independent observers in 7-10 (depending on the specimen size) adjacent high-power fields and graded: 0 (staining not detectable), 1 (weak immunostaining), 2 (moderate immunostaining intensity), or 3 (strong staining). The mean grade was calculated by averaging grades assigned by the two authors and approximating the arithmetical mean to the nearest unity.

Morphometry

CD31-positive vessels were evaluated using a computer image analysis system consisting of a PC equipped with a Pentagram graphic tablet, Indeo Fast card (frame grabber, true-colour, real-time), produced by Indeo (Taiwan), and colour TV camera Panasonic (Japan) coupled with a Carl Zeiss microscope (Germany). This system was programmed (MultiScan 18.03 software, produced by Computer Scanning Systems, Poland) to calculate the number of objects (semiautomatic function).
CD31 immunostains were evaluated in the vessels only (not in the individual cells), in the most vascular areas. The number of CD31-positive vessels (microvessel density) was estimated by counting all positive vessels in 7-10 high-power fields (HPF) (0.25 mm² each), marking immunopositive cells (semiautomatic function). The results are presented as the number of positive vessels per HPF.

Statistical methods

Differences between groups were tested using one-way ANOVA with post-hoc Tukey test. The Kruskal-Wallis test with post-hoc Dunn’s test was used where appropriate. Correlation coefficients were calculated using Spearman’s method. Results were considered statistically significant if p < 0.05.

Results

The immunoexpression of ADAM10 was localised in the epithelial cells, dysplastic and cancer cells, vascular endothelial cells, and infrequently in the infiltrating stromal lymphocytes. Our study revealed that the majority of the ADAM10 was localised in the cytoplasm, but extracellular immunoexpression was also noted.
The semiquantitative data of the immunoexpression of ADAM10 and the number of CD31+ vessels appear in Table I. The immunoexpression of ADAM10 in the OSCC M(+) group (Fig. 1A) was significantly increased in comparison to OSCC M(–) (Fig. 1B), both groups of OLK (Fig. 1C), and control groups (Fig. 1D). Moreover, in the OSCC M(–) group immunoexpression of ADAM10 was significantly increased in comparison to OLK-LG, OLK-HG, and controls. No statistically significant differences were found between immunoexpression of ADAM10 in both groups of OLK and control cases.
The mean number of the vessels was significantly increased in the OSCC M(+) group (Fig. 1E) in comparison to the OSCC M(–) (Fig. 1F), OLK-LG, OLK-HG, and control group. We found also significantly increased mean number of vessels in the OSCC M(–) group compared to both groups of OLK and the control group.
In OSCC M(+) and OSCC M(–) groups there were positive significant correlations between the immunoexpression of ADAM10 and the microvessel density (r = 0.42, p < 0.007; r = 0.35, p < 0.04, respectively), whereas this correlation was not statistically significant in OLK -LG (r = 0.25, p = 0.36 [NS]) and OLK-HG (r = 0.31, p = 0.41 [NS]).
No statistically significant differences were found between immunoexpression of ADAM10 and the mean number of vessels in the OLK-LG and OLK-HG groups (data not shown).
In the control group this correlation was weak and not significant (data not shown).

Discussion

Many studies have shown that members of the ADAM family mediate fundamental biological processes from cellular adhesion to migration. ADAM activity seems to be related to the control of several signalling pathways activated in cancer [4, 5]. For this reason, ADAM family members could be targets for antitumour therapy [23].
Increased expression of ADAM10 has been found in many cancers, playing a pivotal role in the cancer progression and affecting patient prognosis. Our study indicated that ADAM10 immunoexpression was significantly higher in both groups of OSCCs compared with that in controls. Moreover, our study revealed increased ADAM10 immunoexpression in OSCC with metastasis than in the group of cancers without metastases. In uveal melanoma, Caltabiano et al. also demonstrated significantly higher expression of ADAM10 in patients with metastases in comparison to patients without metastases, suggesting that ADAM10 expression is associated with a more rapid metastatic progression [24]. Zhang et al. revealed that overexpression of ADAM10 in hepatocellular carcinoma was significantly associated with tumour metastasis, as well as tumour grade, differentiation, and tumour size [13]. Our results also seem to be in agreement with experimental in vitro findings. Mullooly et al. reported that knockdown of ADAM10 or treatment with the selective inhibitor of ADAM10 decreased cell migration and invasion in a breast cancer cell line [25].
To the best of our knowledge, this is the first study to examine ADAM10 immunoexpression in premalignant lesions such as OLK. In our study, the immunoexpression of ADAM10 in both groups of OLK was significantly decreased in comparison to OSCC groups. Moreover, there were no statistically significant differences between immunoexpression of ADAM10 in OLK and control cases. Similar to our results, Kauttu et al. demonstrated higher expression of ADAM10 in oesophageal adenocarcinoma than in normal control tissues, but ADAM10 expression in premalignant dysplastic epithelium (Barrett’s dysplasia) did not differ from normal oesophageal squamous epithelium [26]. A possible explanation for our results can be the small number of cases, but it is also possible that our findings reflect the situation that the biological action of ADAM10 decreased cell migration, and invasion influenced the advance stages of oral carcinogenesis but had little impact on the early stages of oral carcinogenesis (OLK).
To the best of our knowledge, this is also the first study considering the tumour immunoexpression of ADAM10 and microvessel density. Literature data shows that increased ADAM10-mediated proteolysis correlates with decreased vascular integrity, increased production of cytokines such as TNF-, and increased transmigration of leukocytes [27]. ADAM10 was also identified as a binding partner of VEGFR2. In addition, VEGF induced ADAM10-mediated cleavage of VE-cadherin, which could increase vascular permeability and facilitate migration [28]. Moreover, it was demonstrated that ADAM10 expressed in human atherosclerotic lesions is associated with neovascularisation [27]. In this context, statistically significant correlation between the ADAM10 overexpression and the process of angiogenesis, defined as the increase in microvessel density in both tested groups of cancer, seems be in agreement with previous findings. On the other hand, Caolo et al. documented in mouse retinas that inhibition of ADAM10 induced vascular sprouting and density [29]. Differences concerning the association between ADAM10 and microvessel density in various studies may indicate that the precise role and mechanisms of action of ADAM10 in the process of angiogenesis are still not fully determined.

Conclusions

In conclusion, the present study revealed overexpression of ADAM10 in OSCC, especially in OSCC with metastases. These findings may indicate that ADAM10 could potentially contribute to metastases of oral cancer and suggest that increased angiogenesis may be involved in this process. However, further research is required, to determine the underlying mechanisms by which ADAM10 might play a role in angiogenesis of the oral region.

This work was supported by a grant from the Medical University of Lodz 503/6-038-01/503-61-002.
The authors declare no conflict of interest.

References

1. Shaw RJ, Pace-Balzan A, Butterworth C. Contemporary clinical management of oral squamous cell carcinoma. Periodontol 2011; 57: 89-101.
2. Ferlay J, Soerjomataram I, Ervik M, et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013. http://globocan.iarc.fr, accessed on 03/09/2016.
3. Napier SS, Speight PM. Natural history of potentially malignant oral lesions and conditions: an overview of the literature. J Oral Pathol Med 2008; 37: 1-10.
4. Edwards DR, Handsley MM, Pennington CJ. The ADAM metalloproteinases. Mol Aspects Med 2008; 29: 258-289.
5. Seals DF, Courtneidge SA. The ADAMs family of metalloproteases: Multidomain proteins with multiple functions. Genes Dev 2003; 17: 7-30.
6. Nakamura T, Abe H, Hirata A, et al. ADAM family protein Mde10 is essential for development of spore envelopes in the fission yeast Schizosaccharomyces pombe. Eukaryot Cell 2004; 3: 27-39.
7. Huxley-Jones J, Clarke TK, Beck C, et al. The evolution of the vertebrate metzincins; insights from Ciona intestinalis and Danio rerio. BMC Evol Biol 2007; 7: 63.
8. Blobel CP. ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 2005; 6: 32-43.
9. Klein T, Bischoff R. Active metalloproteases of the A Disintegrin and Metalloprotease (ADAM) family: biological function and structure. J Proteome Res 2011; 10: 17-33.
10. Wu K, Liao M, Liu B, et al. ADAM-17 over-expression in gallbladder carcinoma correlates with poor prognosis of patients. Med Oncol 2011; 28: 475-480.
11. Saftig P, Reiss K. The „A Disintegrin And Metalloproteases” ADAM10 and ADAM17: novel drug targets with therapeutic potential? Eur J Cell Biol 2011; 90: 527-535.
12. Anders L, Mertins P, Lammich S, et al. Furin-, ADAM 10-, and gamma-secretase-mediated cleavage of a receptor tyrosine phosphatase and regulation of beta-catenin’s transcriptional activity. Mol Cell Biol 2006; 26: 3917-3934.
13. Lee SB, Schramme A, Doberstein K, et al. ADAM10 is upregulated in melanoma metastasis compared with primary melanoma. J Invest Dermatol 2010; 130: 763-773.
14. Zhang W, Liu S, Liu K, et al. A disintegrin and metalloprotease (ADAM)10 is highly expressed in hepatocellular carcinoma and is associated with tumour progression. J Int Med Res 2014; 42: 611-618.
15. Gaida MM, Haag N, Günther F, et al. Expression of A disintegrin and metalloprotease 10 in pancreatic carcinoma. Int J Mol Med 2010; 26: 281-288.
16. Guo J, He L, Yuan P, et al. ADAM10 overexpression in human non-small cell lung cancer correlates with cell migration and invasion through the activation of the Notch1 signaling pathway. Oncol Rep 2012; 28: 1709-1718.
17. Wang YY, Ye ZY, Li L, et al. ADAM 10 is associated with gastric cancer progression and prognosis of patients. J Surg Oncol 2011; 103: 116-123.
18. Fu L, Liu N, Han Y, et al. ADAM10 regulates proliferation, invasion, and chemoresistance of bladder cancer cells. Tumour Biol 2014; 35: 9263-9268.
19. Lendeckel U, Kohl J, Arndt M, et al. Increased expression of ADAM family members in human breast cancer and breast cancer cell lines. J Cancer Res Clin Oncol 2005; 131: 41-48.
20. Liu S, Zhang W, Liu K, et al. Silencing ADAM10 inhibits the in vitro and in vivo growth of hepatocellular carcinoma cancer cells. Mol Med Rep 2015; 11: 597-602.
21. Zhang W, Liu S, Liu K, et al. Knockout of ADAM10 enhances sorafenib antitumor activity of hepatocellular carcinoma in vitro and in vivo. Oncol Rep 2014; 32: 1913-1922.
22. Barnes L, Everson JW, Reichart P, et al. World Health Organization Classification of Tumours. Pathology and Genetics Head and Neck Tumours. IARC Press, Lyon 2005; 168-176.
23. Zhou BB, Peyton M, He B, et al. Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer. Cancer Cell 2006; 10: 39-50.
24. Caltabiano R, Puzzo L, Barresi V, et al. ADAM 10 expression in primary uveal melanoma as prognostic factor for risk of metastasis. Pathol Res Pract 2016; 212: 980-987.
25. Mullooly M, McGowan PM, Kennedy SA, et al. ADAM10: a new player in breast cancer progression? Br J Cancer 2015; 113: 945-951.
26. Kauttu T, Mustonen H, Vainionpää S, et al. Disintegrin and metalloproteinases (ADAMs) expression in gastroesophageal reflux disease and in esophageal adenocarcinoma. Clin Transl Oncol 2017; 19: 58-66.
27. Schulz B, Pruessmeyer J, Maretzky T, et al. ADAM10 regulates endothelial permeability and T-Cell transmigration by proteolysis of vascular endothelial cadherin. Circ Res 2008; 102: 1192-1201.
28. Donners MM, Wolfs IM, Olieslagers S, et al. A disintegrin and metalloprotease 10 is a novel mediator of vascular endothelial growth factor-induced endothelial cell function in angiogenesis and is associated with atherosclerosis. Arterioscler Thromb Vasc Biol 2010; 30: 2188-2195.
29. Caolo V, Swennen G, Chalaris A, et al. ADAM10 and ADAM17 have opposite roles during sprouting angiogenesis. Angiogenesis 2015; 18: 13-22.

Address for correspondence

Olga Stasikowska-Kanicka M.Sc., Ph.D.
Department of Nephropathology
Medical University of Lodz
Czechoslowacka 8/10
92-216 Lodz, Poland
tel.fax +48 42 679 01 91
e-mail: olga.stasikowska@umed.lodz.pl