Laboratory Investigation (2017) 97, 1332–1342 © 2017 USCAP, Inc All rights reserved 0023-6837/17 Brain-derived neurotrophic factor/tropomyosin-related kinase B signaling pathway contributes to the aggressive behavior of lung squamous cell carcinoma Keigo Ozono1,2, Yoshihiro Ohishi1, Hideya Onishi3, Katsuya Nakamura3,4, Junichi Motoshita5, Masato Kato6, Ryoichi Nakanishi7, Masafumi Nakamura2 and Yoshinao Oda1 The tropomyosin-related kinase (Trk) family consists of TrkA, TrkB, and TrkC, which play essential roles in tumor progression and/or suppression in various cancers. Little is known about the biological significance of the Trk family in human lung squamous cell carcinoma (SCC). Here we investigated the clinical significance of the protein expression of Trk family members in samples from 99 SCC patients, and we explored the relationship between invasion/proliferation activities and Trk expression using lung SCC cell lines to clarify the biological significance of the Trk family in lung SCC. Immunohistochemical high expression of TrkB was significantly correlated with vascular invasion (P = 0.004), lymph node metastasis (Po0.001), and advanced stage (P = 0.0015). The overall survival of the patients with TrkB-high expression was significantly shorter than those with TrkB-low expression (P = 0.0110). TrkA/TrkC expressions were not predictors of poor prognosis. An in vitro assay demonstrated that the inhibition of brain-derived neurotrophic factor (BDNF) (a TrkB ligand) and TrkB by K252a (a Trk inhibitor) or siRNA (BDNF-siRNA, TrkB-siRNA) suppressed the invasion, migration, and proliferative activities of lung SCC cells. The administration of recombinant human BDNF (rhBDNF) enhanced the invasion, migration, and proliferation activities, which were abrogated by K252a. TrkB-siRNA transfection increased the protein expression of E-cadherin and decreased vimentin expressions in lung SCC cells. Matrix metalloproteinase-2 (MMP-2)- mediated gelatin degradations were decreased in lung SCC cells transfected with TrkB-siRNA. Thus, TrkB-high expression is an indicator of poor prognosis in lung SCC, probably due to invasion/proliferation activities promoted by the BDNF/TrkB signaling pathway, which could become a therapeutic target for lung SCC. Laboratory Investigation (2017) 97, 1332–1342; doi:10.1038/labinvest.2017.45; published online 12 June 2017 Non-small-cell lung cancer (NSCLC) accounts for ~ 80% of subset of adenocarcinomas.7,8 Molecular target therapy for lung cancers and remains the most common cause of cancer- advanced SCC of the lung is not yet well established. The related deaths worldwide.1 Squamous cell carcinoma (SCC) is development of new molecular target therapies for advanced the second most common subtype of NSCLC (adenocarci- SCC of the lung is thus a pressing issue. noma is the first), accounting for ~ 20–30% of NSCLC cases.2 Members of the tropomyosin-related kinase (Trk) family The treatment for a subset of lung adenocarcinomas has are tyrosine kinase receptors: TrkA (NTRK1), TrkB greatly improved due to the development of therapies using (NTRK2), and TrkC (NTRK3). Primary ligands with high the epidermal growth factor receptor (EGFR) tyrosine kinase affinity are nerve growth factor (NGF) for TrkA, brain- inhibitors gefitinib and erlotinib3–6 or an anaplastic lym- derived neurotrophic factor (BDNF) for TrkB, and phoma kinase (ALK) inhibitor, crizotinib.7,8 neurotrophin-3 (NT-3) for TrkC.14,15 The Trk family The efficacy of EGFR tyrosine kinase inhibitor (TKI) regulates the development of the nervous system and neural against lung SCC remains controversial,9–13 but an ALK TKI origin cells, and Trk family members are involved in the has been demonstrated to be effective, especially for a small maintenance of neural tissue.14–17 1Department of Anatomic Pathology, Kyushu University, Fukuoka, Japan; 2Department of Surgery and Oncology, Kyushu University, Fukuoka, Japan; 3Department of Cancer Therapy and Research, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 4Department of Thoracic Surgery, Japan Community Health Care Organization, Kyushu Hospital, Fukuoka, Japan; 5Department of Diagnostic Pathology, Hamanomachi Hospital, Fukuoka, Japan; 6Department of Surgery, Hiramatsu Hospital, Saga, Japan and 7Department of Oncology, Immunology and Surgery, Graduate School of Medical Sciences and Medical School, Nagoya City University, Nagoya, Japan Correspondence: Professor Y Oda, PhD, Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: [email protected] Received 25 September 2016; revised 14 March 2017; accepted 21 March 2017 1332 Laboratory Investigation | Volume 97 November 2017 | www.laboratoryinvestigation.org BDNF/TrkB contributes to the aggressive behavior K Ozono et al Many studies have indicated that Trk family members also have oncogenic functions in several malignant tumors.18–44 Interestingly, the oncological roles of TrkA, TrkB, and TrkC are different among tumor types despite their close homology. For example, TrkA overexpression is associated with poor prognosis in thyroid carcinoma,18 breast cancer,19 and ovarian serous carcinoma,20 whereas TrkA acts as a tumor suppressor in neuroblastoma21 and colon cancer22 as shown in in vitro assays. TrkB overexpression was reported to act as an oncogenic molecule in pancreas cancer,23 hepatocellular cancer,24 prostate cancer,25 ovarian cancer,26 Wilm’s tumor,27 gastric cancer,28,29 breast cancer,30 colon cancer,31–33 oral squamous cell carcinoma,34 head and neck squamous cell carcinoma,35 and neuroblastoma.36 TrkC overexpression is Figure 1 The role of the BDNF/TrkB signaling pathway in lung SCC. The BDNF/TrkB signaling pathway enhances the cell proliferation and invasion reported as an indicator of good prognosis for activity via migration, the EMT and MMP-2 activation. medulloblastoma37 and colon cancer.38 With regard to lung cancer, Ricci et al. reported constant BDNF and TrkB expressions in human lung SCC samples.39 All of the surgical specimens were routinely fixed by Higher TrkB expression in NSCLC including SCC was formalin before the histopathological and immunohisto- reported to be correlated with lymph node metastasis40 and chemical evaluations. The patients' clinicopathological pro- vascular invasion.41 Okamura et al. reported that high TrkB files are shown in Table 1. The histological subtypes of the expression was correlated with short periods of disease-free tumors and the pathological stage were classified according to and overall survival in NSCLC patients.41 In addition, Terry the World Health Organization (WHO) classification of lung et al. reported that positive TrkB staining was correlated with and pleural tumors,45 and the Union for International Cancer improved disease-specific survival and overall survival in lung Control (UICC) guidelines for tumor, node, metastasis SCC.42 (TNM) classification.46 In terms of the function of the BDNF/TrkB signaling This study was approved by the Ethics Committee of pathway, it was reported that enhancement of the BDNF/ Kyushu University (No. 28-71). TrkB pathway induced invasion activity in pulmonary large cell neuroendocrine carcinoma (LCNEC) cells43 and lung Immunohistochemistry adenocarcinoma cells.40 The Trk inhibitor K252a was Immunohistochemical staining (IHC) was performed using reported to inhibit the invasion activity of LCNEC cells,43 4-μm-thick formalin-fixed, paraffin-embedded tissue sections induce cell death,44 and diminish the colony formation of and the primary antibodies TrkA (sc-118, Santa Cruz adenocarcinoma cells.44 However, a functional analysis of the Biotechnology, Santa Cruz, CA, USA, 1:50), TrkB (sc-8316, BDNF/TrkB signaling pathway of lung SCC has not been Santa Cruz Biotechnology, 1:50) and TrkC (ab77494, Abcam, established. Cambridge, UK, 1:200). Endogenous peroxidase activity was In this study, we hypothesized that BDNF/TrkB signal blocked for 30 min by methanol containing 0.3% hydrogen promotes proliferating, migratory and invasive phenotypes peroxidase. For TrkA and TrkB, sections were incubated with and cellular plasticity (eg, epithelial–mesenchymal transition primary antibodies overnight at 4 °C, followed by a 40-min [EMT] and MMP level) in lung SCC (Figure 1). Accordingly, incubation with the secondary antibodies at room tempera- we investigated43 the clinical significance of the protein ture. For TrkC, antigen retrieval was conducted by microwave expression of Trk family members using clinical samples of heating for 20 min with target retrieval solution (TRS) buffer. lung SCC, and we examined the relationship between the TrkC antibody incubation was followed by standard signal BDNF/TrkB signaling pathway and the invasion/migration amplification, including rabbit LINKER (K8019) at room and proliferation activities of lung SCC cells. temperature for 15 min, and then horseradish peroxidase (HRP)-conjugated EnVision FLEX (Dako, Glostrup, Den- MATERIALS AND METHODS mark) at room temperature for 20 min. The reaction products Patients and Case Selection were visualized by diaminobenzidine (DAB). We retrospectively analyzed the cases of 99 patients with lung We evaluated the proportion and intensity of the SCC who underwent curative surgical resection at the immunoreactive cells on the entire slides by using the Allred Department of Surgery and Oncology, Kyushu University score (AS) as described.32 The proportional score (PS) was Hospital, the Department