Abstract
Background/Aim: Thymic epithelial tumors (TETs) mainly consist of thymoma and thymic carcinoma. Complete surgical resection is vital for the successful management of these TETs, and adjuvant therapy such as systematic chemotherapy and/or radiotherapy plays important roles in the management of recurrent or metastasized disease. However, there is still a lack of a standard treatment after the failure of these adjuvant therapies. There is thus a need to develop molecular targeted therapies for advanced malignant TETs. In the present study, we evaluated the biological significance of brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signaling for TETs. Materials and Methods: The expression of TrkB in 48 formalin-fixed, paraffin-embedded TET specimens (43 thymoma and 5 thymic carcinoma) collected by surgical resection was evaluated immunohistochemically. A thymic carcinoma cell line was evaluated for the role of BDNF/TrkB signaling pathway in an in vitro assay. Results: High TrkB expression was related to significantly poor prognosis in patients with TETs. In vitro experiments showed that BDNF/TrkB signaling was involved in the proliferation of Ty-82 cells, but not their invasion and migration. Conclusion: TrkB expression is a biomarker of the prognosis for TETs and the BDNF/TrkB signaling pathway is potentially a new therapeutic target for mTETs.
Thymic epithelial tumors (TETs) are the most common of all adult tumors arising in the mediastinum (1). The TETs include thymoma, which is rare, with 1.5 cases occurring per million, and thymic carcinoma, which is very rare (2). Histologically, thymoma is divided into different entities: Types A, AB, B1, B2, and B3 (3). Clinically, regarding malignancy, TETs are divided into two groups: low grade TETs (lTETs) as Type A, AB, and B1 thymoma and high grade TETs (hTETs) as Type B2 and B3 thymoma, and thymic carcinoma. hTETs often show aggressive behavior and can recur or metastasize (4, 5).
Complete surgical resection is vital for the successful management of these TETs, and adjuvant therapy such as systematic chemotherapy and/or radiotherapy plays important roles in the management of recurrent or metastasized disease. However, there is still a lack of a standard treatment after the failure of these adjuvant therapies (6). The search for biologic agents for treating thymoma and thymic carcinoma has thus far yielded disappointing results (7-10). There is thus a need to develop molecular targeted therapies for advanced hTETs.
The tropomyosin receptor kinase (Trk) family is a group of tyrosine kinase receptors consisting of TrkA (NTRK1), TrkB (NTRK2), and TrkC (NTRK3). Primary ligands with high affinity for members of this family include nerve growth factor (NGF) for TrkA, brain-derived neurotrophic factor (BDNF) for TrkB, and neurotrophin-3 (NT-3) for TrkC (11, 12). The Trk family regulates development of the nervous system and neuron-derived cells, and is involved in the maintenance of neural tissue (11-14). The Trk family members also have oncogenic functions in various tumors (15-41). For example, TrkB has been reported to be a poor prognostic and oncogenic factor in pancreatic cancer (20), hepatocellular cancer (21), prostate cancer (22), ovarian cancer (23), Wilms’ tumor (24), gastric cancer (25, 26), breast cancer (27), colon cancer (28-30), head and neck squamous cell carcinoma (31), neuroblastoma (32), and pulmonary large cell neuroendocrine carcinoma (LCNEC) (33).
Previously, we showed that BDNF/Trk B signaling contributes to inducing a malignant phenotype in lung squamous cell carcinoma and that it could be a new therapeutic target (34). In the present study, we analyzed the biological significance of BDNF/TrkB signaling in TETs.
Materials and Methods
Patients and case selection. We examined retrospectively 48 patients with thymoma and thymic carcinoma (43 with thymoma and 5 with thymic carcinoma) who underwent curative surgical resection and diagnosed pathologically at Kyushu University Hospital. The surgically resected specimens were routinely fixed in formalin and subjected to histopathological processing and immunohistochemical evaluation. The clinicopathological profiles including age, sex, and Masaoka-Koga stage are shown in Table I. Regarding malignancy, we divided the TETs into two groups: low grade TETs (lTETs) as Type A, AB, and B1 thymoma, and high grade TETs (hTETs) as Type B2 and B3 thymoma, and thymic carcinoma. We also divided thymoma and thymic carcinoma into two groups based on the Masaoka-Koga stage: We defined stages I and II as early stage and stages III and IV as advanced stage.
Clinicopathological features and immunohistochemical TrkA, TrkB, and TrkC expressions of patients with thymic epithelial tumors.
Histological subtypes of the tumors were classified according to the World Health Organization (WHO) classification of Tumors of the Lung, Pleura, Thymus, and Heart 4th Edition (42). This study was approved by the Ethics Committee of Kyushu University (No. 28-71) and conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from the patients to participate and publication of this research.
Immunohistochemistry. Immunohistochemical (IHC) staining was performed as described previously (34). In brief, 4-μm-thick sections were obtained from formalin-fixed tissues and IHC was performed using primary antibodies, TrkA (1:50; Santa Cruz Biotechnology, Dallas, TX, USA), TrkB (1:50; Santa Cruz Biotechnology), and TrkC (1:200; Abcam, Cambridge, UK).
IHC analysis was performed following the Allred score (AS). The proportional score (PS) was defined as: 0%=PS0, 0% < PS1≤1%, 1%<PS2≤10%, 10%<PS3≤33%, 33%<PS4 ≤67%, and 67%<PS5 ≤100%, whereas the intensity score (IS) was defined as: IS0=negative, IS1=weak, IS2=intermediate, and IS3=strong. PS and IS were summed up for obtaining a total score (TS) (0, 2 to 8) and divided into four grades: Grade 0, TS is 0; Grade 1, TS is 2-4; Grade 2, TS is 5-6, and Grade 3, TS is 7-8. Grades 2 and 3 were regarded as reflecting high expression.
Cell line. A human thymic carcinoma cell line Ty-82 was obtained from JCRB (Tsukuba, Japan) and maintained in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS).
Western blot analysis. Western blotting was performed as described previously (34). In brief, proteins were extracted using PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Sungnam, Republic of Korea) and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies at the recommended dilution [TrkB (1:250), BDNF (1:250), GAPDH (6C5, 1:500; Santa Cruz Biotechnology), MMP-2 (1:200; Santa Cruz Biotechnology), MMP-9 (1:200; Santa Cruz Biotechnology), E-cadherin (36/E-Cadherin, 1:2,500; BD Transduction Laboratories, Franklin Lakes, NJ, USA), and vimentin (V9, 1:200)].
Anchorage-dependent cell proliferation assay. Cell proliferation was determined by using the Trk inhibitor K252a (Alomone Labs, Jerusalem, Israel), recombinant human BDNF (rhBDNF) (Peprotech, Rocky Hill, NJ, USA), and siRNA (Control-siRNA, TrkB-siRNA, and BDNF-siRNA). The Ty-82 cell line (2×103 cells/well) was incubated in each well of 96-well culture plate with the medium described above for 48 h. At regular intervals (0, 24, and 48 h), we measured the optical density at 492 nm (ref. 620 nm) using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). The cell survival rate is expressed as the absorbance. The experiments were performed three times in triplicate wells.
Cell invasion assays. Cell invasion assay was performed using siRNA (Control-siRNA, TrkB-siRNA, and BDNF-siRNA). Biocoat Matrigel invasion chambers (BD Biosciences, San Diego, CA, USA) were used to evaluate cell invasion, in accordance with the manufacturer’s protocol. In brief, the cells (1×105) were detached and seeded in the upper chamber of an 8-μm pore-size insert pre-coated with Matrigel. The outer wells were filled with medium containing 5% FBS.
After 24 h of incubation at 37°C with 5% carbon dioxide, non-invading cells on the upper membrane surface were removed with a cotton tip. Invading cells attached to the lower surface of the Transwell membrane with 70% ethanol and stained with hematoxylin-eosin. The membrane was cut and laid on a microscope slide, and the number of invading cells was counted in five randomly-selected high-powered fields.
Cell migration assays. Cell migration assay was performed using siRNA (Control-siRNA, TrkB-siRNA, and BDNF-siRNA). Falcon Cell Culture Inserts (Thermo Fisher Scientific, Pittsburgh, PA, USA) was used to assess the migration assay. Cells were detached and 1×105 cells were seeded on top of an 8-μm pore-size filter in the upper chamber in serum-free medium. The bottom of each pore contained a polyethylene terephthalate membrane. The cells were incubated for 16 h at 37°C with 5% carbon dioxide. Non-migrating cells were removed by a cotton tip and migrating cells were fixed to the lower surface of the membrane with 70% ethanol and stained with hematoxylin-eosin. The membrane was cut and laid on a microscope slide, and migrating cells were counted in five randomly selected high-powered fields.
Gelatin zymography. We performed gelatin zymography for assessing MMP enzyme activity according to the manufacturer’s instructions. Briefly, TrkB-siRNA was transfected into cells. After incubated for 48 h, protein samples were extracted and analyzed using a Gelatin Zymography Kit (Primary Cell, Ishikari, Japan). Areas of gelatin degraded by MMP were visualized as a clear zone against a blue background.
Statistical analysis. All statistical analyses were performed using JMP Statistical Discovery Software (version 11.0; SAS, Cary, NC, USA). All data are presented as mean±standard deviation (SD). Relationship between clinicopathological findings and biomarkers were evaluated by using Fisher’s exact test. Actuarial survival curves were obtained using the Kaplan–Meier method, and comparisons were made using the log-rank test. p<0.05 was considered statistically significant.
Results
Trk family expression in thymoma and thymic carcinoma. The characteristics of all 48 patients with thymoma and thymic carcinoma (43 with thymoma and 5 with thymic carcinoma) who underwent curative surgical resection are summarized in Table I. They ranged in age from 29 to 80 (median 56.2) years old. Twenty-two were male and 26 were female. With regard to the Masaoka-Koga stage, there were 37 cases (77%) in early stage (Stages I and II) and 11 cases (22%) in advanced stage (Stages III and IV). Regarding malignancy, low grade TETs (lTETs; Type A, AB, and B1 thymoma) numbered 29 cases (60%) and high grade TETs (hTETs; Type B2 and B3 thymoma, and thymic carcinoma) numbered 19 cases (39%).
The results of IHC analysis of the expression of TrkA, TrkB, and TrkC in 43 thymoma and 5 thymic carcinoma patients are summarized in Table I. In 48 tumor samples, the high expression of TrkA was observed in five cases (10%) and that of TrkB in seven cases (14%). TrkC expression was absent in all 48 cases (0%).
Among the TrkB-high cases, all five thymic carcinoma cases exhibited high TrkB expression (Figure 1A-E); the other two cases were type B1 (Figure 1F) thymoma and type B2 thymoma (Figure 1G and H). This latter case of TrkB-high type B2 thymoma involved recurrence. There was no correlation between Trk expression and clinicopathological factors (age, sex) (Table I).
Trk immunohistochemical expression in clinical samples of thymic carcinomas and thymomas. The five thymic carcinomas present high expression of TrkB. (A-E) Cases 1-5, respectively (200×). High immunohistochemical expression of TrkB in thymomas. (F) Case 6, a type B3 thymoma (200×). (G) and (H) Case 7, a type B2 thymoma: (G) is a sample from the initial surgery (200×) and (H) is one upon recurrence (200×).
High TrkB expression is significantly related to poor prognosis in patients with TETs. With regard to the Masaoka-Koga stage, cases at an advanced stage showed significantly higher expression of TrkB than those at an early stage (p<0.0001), while TrkA and TrkC expression showed no correlation with Masaoka-Koga stage (TrkA: p=0.5163) (Table I). With regard to malignancy, when comparing lTETs and hTETs, there were significantly more cases with high TrkB expression among hTETs than among lTETs (p=0.0004) (Table I), whereas high TrkA expression did not show any correlation with malignancy (p=0.9839).
Information about the survival period was available for all but 2 of the 48 cases. Cases with high TrkB expression had a significantly shorter overall survival period (p=0.0465) compared to those with low expression (Figure 2A). There was no significant correlation between TrkA expression and survival period (p=0.1484) (Figure 2B).
Kaplan–Meier survival curves for TET patients with TrkA, B-high or -low tumors. (A) Patients with TrkB-high tumors have poor overall survival compared with those with TrkB-low tumors. (B) There was no significant difference of overall survival between patients with TrkA-high and -low tumors. p-Values calculated by the log-rank test are indicated. *p<0.05.
From these immunohistochemical staining results, high TrkB expression might raise concerns about malignant potential and could be a biomarker of poor prognosis of thymic epithelial tumors.
Inhibition of BDNF/TrkB signaling suppresses the proliferative ability of thymic carcinoma cells. We examined the role of the BDNF/TrkB signaling pathway in thymic carcinoma by performing an in vitro assay to substantiate the clinical significance of TrkB expression as demonstrated by immunohistochemical staining. We performed western blot analysis to reveal the expression of endogenous TrkB protein and its ligand BDNF in the human thymic carcinoma cell line Ty-82. The cells showed endogenous expression of both BDNF and TrkB (Figure 3A). Western blotting also confirmed the down-regulation of TrkB and BDNF proteins extracted from the cells transfected with TrkB-siRNA and BDNF-siRNA, respectively (Figure 3B).
Protein lysates extracted from thymic carcinoma cells (Ty-82) were subjected to western blot analysis for TrkB and BDNF. (A) The Ty-82 cells showed endogenous TrkB and BDNF expression. Western blotting showed (B) decreased levels of TrkB and BDNF proteins in cells transfected with TrkB-siRNA and BDNF-siRNA, compared with the levels with Control-siRNA.
To assess the effect of the BDNF/TrkB signaling pathway on proliferation, we performed a proliferation assay. Ty-82 cells transfected with BDNF-siRNA or TrkB-siRNA showed significantly reduced proliferation compared to those with Control-siRNA, while the administration of 10 ng/ml rhBDNF to the culture medium of Ty-82 cells transfected with TrkB-siRNA did not lead to a rise in the rate of proliferation (Figure 4A). These findings indicate that the knockdown of BDNF or TrkB suppressed proliferation.
The BDNF/TrkB pathway is involved in proliferation. (a) Ty-82 cells transfected with TrkB-siRNA or BDNF-siRNA showed decreased proliferation compared with those transfected with Control-siRNA. Administering rhBDNF to the cells transfected with TrkB-siRNA showed no increase in proliferation. *p<0.05.
Ty-82 cells incubated with 10 ng/ml rhBDNF showed significantly higher proliferation than those incubated without rhBDNF. Meanwhile, the administration of 50 nM K252a and 10 ng/ml rhBDNF to the culture medium of Ty-82 cells resulted in decreased proliferation compared to that without the administration of K252a (Figure 4B). These results indicated that the proliferative ability was enhanced by rhBDNF and abrogated by the Trk inhibitor K252a.
Inhibition of BDNF/TrkB signaling did not suppress the invasion and migration of thymic carcinoma cells. To assess whether the BDNF/TrkB signaling pathway participates in the invasiveness of thymic carcinoma, we performed invasion and migration assays. Thymic carcinoma Ty-82 cells showed no significant changes in invasion and migration abilities upon knockdown of BDNF or TrkB by using siRNA compared with the case for cells transfected with Control-siRNA (Figure 5A and B). These results indicate that the BDNF/TrkB signaling pathway is not involved in the invasion and migration abilities of thymic carcinoma cells.
TrkB is not involved in the invasiveness of Ty-82 cells. Ty-82 cells transfected with BDNF-siRNA or TrkB-siRNA showed no difference compared with cells transfected with Control-siRNA in terms of (A) invasion and (B) migration abilities. (C) Western blotting revealed that Ty-82 cells transfected with TrkB-siRNA showed no increase in the expression of E-cadherin protein and no decrease in the expression of vimentin protein, and showed no difference in either MMP-2 or MMP-9 expression compared with the cells transfected with Control-siRNA. (D) Gelatin zymography demonstrated no significant difference in gelatin degradation of either MMP-2 or MMP-9 in the culture medium of Ty-82 cells transfected with TrkB-siRNA compared with those transfected with Control-siRNA.
To confirm that the BDNF/TrkB signaling pathway does not participate in the invasiveness, we performed western blot analysis of epithelial-mesenchymal transition (EMT), which is one feature mediating invasiveness. The Ty-82 cell line transfected with TrkB-siRNA showed no increase in E-cadherin protein expression and no decrease in vimentin protein expression compared with the cells transfected with Control-siRNA (Figure 5C), indicating that BDNF/TrkB is not involved in EMT.
We also determined whether the BDNF/TrkB signaling pathway influenced members of the matrix metalloproteinase family, such as matrix metalloproteinase (MMP)-2 and MMP-9, which are other components mediating invasiveness, by performing western blot analysis and gelatin zymography. Western blotting showed no significant difference in either MMP-2 or MMP-9 protein expression in cells transfected with TrkB-siRNA compared with that in cells transfected with Control-siRNA (Figure 5C).
Gelatin zymography demonstrated no significant difference in gelatin degradation of either MMP-2 or MMP-9 in the culture medium of Ty-82 cells transfected with TrkB-siRNA compared with that in cells transfected with Control-siRNA (Figure 5D). These results indicate that the BDNF/TrkB signaling pathway does not influence the MMP family.
Discussion
This study analyzed the behavior of Trk family members and whether they could be therapeutic targets for hTETs. We have previously shown that the BDNF/TrkB signaling pathway plays an important role in tumor aggressiveness due to its effects on invasiveness and proliferation in lung squamous cell carcinoma and that this pathway could be a therapeutic target (34). Immunohistochemical staining results showed that thymic carcinoma expressed TrkB. Interestingly, among the two cases of TrkB-high thymoma, one was type B3 and the other was a recurrent case of type B2, indicating that TrkB expression correlated with the aggressiveness of TETs. The number of cases with high TrkB expression was significantly greater in advanced cases than in those at an early stage. In addition, the number of cases with high TrkB expression was greater in malignant cases than in benign ones. With regard to the survival period, cases with high TrkB expression had significantly shorter overall survival compared with those with low expression, indicating that TrkB expression could be a biomarker of poor prognosis in TET patients.
However, we should point out that conflicting results have been reported showing that, immunohistochemically, thymic epithelial tumors do not express TrkB (43). This discrepancy could be explained by a different method being used for staining and evaluating the expression levels in that study, as no standard method for this has yet been established. Our immunohistochemical staining results are in line with those of many other studies demonstrating that TrkB is an indicator of poor prognosis in malignant tumors (20-34, 37-41). There are reports describing that TrkA is both a favorable and an unfavorable factor in various tumors (15-19). Meanwhile, TrkC has been reported as a favorable factor in malignant tumors (35, 36). In this study, no correlations of TrkA and TrkC expression with clinicopathological factors or survival period were identified, suggesting that TrkA and TrkC are not involved in the biological behavior of TETs.
Based on our observation that TrkB expression is an indicator of poor prognosis in thymoma and thymic carcinoma samples, we investigated the biological effects of the BDNF/TrkB signaling pathway on thymic carcinoma cells in vitro. The results of an anchorage-dependent proliferation assay indicated that the BDNF/TrkB pathway was involved in the proliferation of thymic carcinoma cells, which was impeded by the inhibition of this pathway; this indicated that BDNF/TrkB could be a therapeutic target. Indeed, several reports have described that the BDNF/TrkB signaling pathway is involved in the proliferation of various different malignant cells (21, 25, 28, 30, 32, 34, 41), in line with our results. However, conflicting results have been reported that the BDNF/TrkB pathway is not involved in the proliferation of LCNEC cells (33). We thus consider that the effect of the BDNF/TrkB signaling pathway on cell proliferation may differ among tumor types. Unexpectedly, our findings showed that the BDNF/TrkB signaling pathway is not involved in the invasiveness of thymic carcinoma cells.
This is the first study demonstrating that the expression of TrkB protein could be a biomarker of poor prognosis of thymic epithelial tumors by using clinical samples. It is also the first study showing that the BDNF/TrkB signaling pathway is involved in proliferation and could be inhibited by Trk inhibitor, as revealed via an in vitro assay. These results may pave the way for a targeted therapy for thymic carcinoma and show that Trk inhibitor could be a therapeutic option for patients with treatment-resistant hTETs.
In conclusion, our results strongly suggest that TrkB expression is a biomarker for the prognosis for TETs and that the BDNF/TrkB signaling pathway is potentially a new therapeutic target for hTETs.
Acknowledgements
The Authors thank Dr. Makoto Kawamoto, Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan for his cooperation.
Footnotes
Authors’ Contributions
Keigo Ozono participated in the design of the study and drafted the manuscript. Naoya Iwamoto, Katsuya Nakamura, and Kei Miyoshi participated in technical and material support. Hideya Onishi, Masafumi Nakamura, and Yoshinao Oda participated in the supervision of the study and helped to draft the manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors have no competing interests to declare in relation to this study.
- Received April 26, 2022.
- Revision received June 14, 2022.
- Accepted June 24, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
