1856 ONCOLOGY LETTERS 19: 1856-1864, 2020 ADAM9: A novel player in vestibular schwannoma pathogenesis MARIA BREUN1, ALEXANDRA SCHWERDTFEGER1, DONATO DANIEL MARTELLOTTA1, ALMUTH F. KESSLER1, CAMELIA M. MONORANU2, CORDULA MATTHIES1, MARIO LÖHR1* and CARSTEN HAGEMANN1* 1Department of Neurosurgery, University Hospital Würzburg; 2Department of Neuropathology, Institute of Pathology, University of Würzburg, D-97080 Würzburg, Germany Received April 27, 2019; Accepted October 2, 2019 DOI: 10.3892/ol.2020.11299 Abstract. A disintegrin and metalloproteinase 9 (ADAM9) is VS samples (n=60). A total of 30 of them were from patients a member of the transmembrane ADAM family. It is expressed with neurofibromatosis. Healthy peripheral nerves from in different types of solid cancer and promotes tumor invasive- autopsies (n=10) served as controls. ADAM9 mRNA levels ness. To the best of our knowledge, the present study was the were measured by PCR, and protein levels were determined first to examine ADAM9 expression in vestibular schwan- by immunohistochemistry (IHC) and western blotting (WB). nomas (VS) from patients with and without neurofibromatosis The Hannover Classification was used to categorize tumor type 2 (NF2) and to associate the data with clinical parameters extension and hearing loss. ADAM9 mRNA levels were of the patients. The aim of the present study was to evaluate 8.8-fold higher in VS compared with in controls. The levels if ADAM9 could be used as prognostic marker or therapeutic were 5.6-fold higher in patients with NF2 and 12-fold higher in target. ADAM9 mRNA and protein levels were measured in patients with sporadic VS. WB revealed two mature isoforms of the protein, and according to IHC ADAM9 was mainly expressed by S100-positive Schwann cells. There was a strong correlation between ADAM9 mRNA expression and the level of functional impairment (r~1, p=0.01). Particularly, the secreted Correspondence to: Dr Maria Breun, Department of Neurosurgery, University Hospital Würzburg, Josef-Schneider-Straße 11, isoform of ADAM9 was expressed in patients with higher D-97080 Würzburg, Germany hearing impairment. ADAM9 mRNA was overexpressed in the E-mail: [email protected] tumor samples relative to healthy vestibular nerves, and there was an association between higher ADAM9 expression levels Abbreviations: ADAM, a disintegrin and metalloproteinase; Akt, and greater hearing impairment. Therefore, ADAM9 may be a protein kinase B; bp, base pair; CCC, comprehensive cancer center; prognostic marker for VS, and ADAM9 inhibition might have CD44, cell adhesion molecule 44; CD68, cell adhesion molecule 68; the potential as a systemic approach for the treatment of VS. cDNA, complementary desoxyribonucleic acid; Cy2/3, cyanine 2/3; dB, decibel; ECL, enhanced chemiluminescence; EGF, epidermal growth Introduction factor; Erb/Her4, erythroblastic oncogene; FAM, fluorescein amidite; FERM, 4.1 ezrin/radixin/moesin protein; FGF-R, fibroblast growth Vestibular schwannomas (VS) are benign nerve sheath tumors factor receptor; Grb, growth factor receptor-bound protein; HRP, horseradish peroxidase; Hx, hearing class according to the Hannover of the vestibular nerve that arise from neoplastic Schwann Classification; IgG, immunoglobulin G; IHC, immunohistochemistry; cells (1,2). These tumors usually appear sporadically, but IP3, inositol trisphosphate; MAP/MEK, mitogen-activated ERK in rare cases (1:33,000) they are part of a genetic disorder, kinase; MMP, matrix metalloproteinase; NF2, neurofibromatosis; called neurofibromatosis type 2. NF2 is associated with the PI3, phosphoinositide 3-kinase; proHB-EGF, pro-heparine binding loss of the NF2 gene on chromosome 22, which encodes for epidermal growth factor; PKC, protein kinase C; qPCR, quantitative merlin, a tumor suppressor protein (3,4). NF2 patients develop polymerase chain reaction; r.p., rapidly progressive; S100, Schwann cell a multitude of tumors like meningeomas, ependymomas and marker protein 100; SH3, src homology 3; Src, sarcoma; s.p., slowly as the hallmark tumors bilateral VS. These tumors usually progressive; TIMP, tissue inhibitor of matrix metalloproteinase; TNFα, have a higher recurrence rate, grow faster, and are much more tissue necrosis factor α; Tx, tumor extension according to the Hannover adherent to the surrounding structures compared to their Classification; VEGF, vascular endothelial growth factor; VIC, sporadic counterparts (5). Therefore, they are often associated 2'-chloro-7'phenyl-1,4-dichloro-6-carboxy-fluorescein; VS, Vestibular schwannoma; WB, western blotting with persistent cranial nerve deficits and sole surgery is not a long-lasting solution in these cases-in contrast to sporadic VS. *Contributed equally Thus, an efficacious systemic therapy is urgently needed. The main known pathomechanism for vestibular schwan- Key words: vestibular schwannoma, a disintegrin and metalloproteinase noma is the loss of function by Merlin. Merlin is a 4.1 9, pathogenesis, molecular marker protein/ezrin/radixin/moesin protein (FERM) that connects the cytoskeleton with the cell membrane. It is activated by the cells' attachment to the extracellular matrix and by BREUN et al: A NOVEL PLAYER IN VESTIBULAR SCHWANNOMA PATHOGENESIS 1857 intercellular adhesion (6). Merlin's loss of function is the main and NF2- associated VS tissue and its correlation to clinical known mechanism for the development of VS and results parameters of the patients, especially their hearing-loss. in the activation of two signaling pathways. These are the Ras/Raf/MEK pathway and the PI3K/Akt/mTOR pathway, Materials and methods which inhibit apoptosis and result in higher cell survival or proliferation rates (3,7,8). Furthermore, the Hippo pathway Tissue samples. The study was approved by the local ethics and the VEGF-mediated signaling pathway, are also activated committee of the University Hospital Würzburg with the by Merlin's loss of function (3,6). Indeed, the VEGF-inhibitor statement number 145/16 and written informed consent was Bevacizumab has been shown to effectively target VEGF obtained from the patients for the use of their tissue. Tissue overexpression in individual cases of NF2 associated VS (3), collection was from 2010-2015. Directly after surgical but only for a short period in the majority of patients. To date, excision, the tissue was divided in half: one half was cryo- there is no effective systemic treatment option available for preserved, and the other half was fixed in formalin. All tumor VS in terms of maintaining a stable disease state or even samples were neuropathologically assessed according to inducing tumor shrinkage. Therefore, there is a huge necessity WHO-criteria (2). Typical for Antoni type A tissue is the high to identify useful molecular therapeutic targets, especially for cell density in a spindle-shaped arrangement, and for Antoni patients with NF2 (3,9). type B tissue a loose meshwork of gelatinous and microcystic Members of the A-Disintegrin and Metalloproteinase tissue is specific (25). Mixed types, called Antoni A/B, are protein (ADAM) family are therapeutic targets for many tumor also common. As controls served 6 normal vestibular nerves entities. The ADAM protein family consists of 21 functional that were obtained from autopsies within the first 24 h after transmembrane proteins with 8 transmembrane domains. death and 4 sural nerves from biopsies. These include a signal peptide, a propeptide, metallopro- teinase activity, a disintegrin sequence, a cysteine-rich region, Clinical parameters. Hearing function and tumor extension an EGF-like domain, a transmembrane part and a cytoplasmic were categorized using the Hannover Classification (26,27). tail (10,11). One member of this protein family is ADAM9, Tone and speech audiometry were measured several days which is encoded on chromosome 8 and was first described in before surgery to estimate hearing function. Hearing deterio- 1996 (12,13). It plays a role in cell-adhesion and cell-signaling ration was categorized into 6 classes in 20 dB steps: H1 has and is overexpressed in several cancers like breast-, non-small a maximum of 20 dB hearing loss in tone audiometry and a cell lung-, pancreas-, stomach-, hepatocellular-cancer, mela- similar good result in speech audiometry, which is a nearly noma and glioblastoma (10,14-17). normal hearing function. H6 means deafness, with at least Two mature isoforms are produced by alternative mRNA 100 dB hearing loss and no speech discrimination (Table I). splicing, a shorter, secreted and a larger, transmembrane The tumor extension into the cerebellopontine angle was version. The first one derives from a 68 kDa precursor and has estimated as follows: T1 is an intrameatal tumor, T2 is an a mature size of 47 kDa while the latter is represented by a intra‑ and extrameatal tumor, a T3A tumor fills the cerebel- 114 kDa precursor and matures as 84 kDa (11,18,19). Functional lopontine cistern, a T3B tumor has contact to the brainstem, differences of these isoforms are subject of recent research. a T4A tumor compresses the brainstem, and a T4B tumor Especially the secreted isoform promotes cell migration and is already dislocates the brain stem (Table II). correlated with a higher tumor invasiveness by modulating the Tumor growth dynamics were categorized by magnetic interaction with integrins (10,11,14,18). One further function of resonance imaging (MRI) performed during a ‘watch and ADAM9 is protein-shedding of proHB-EGF by its proteinase wait’ period before surgery. The histological proliferation