Neurotrophin receptor TrkB promotes lung adenocarcinoma metastasis

Abstract

Lung cancer is notorious for its ability to metastasize, but the pathways regulating lung cancer metastasis are largely unknown. An in vitro system designed to discover factors critical for lung cancer cell migration identified brain-derived neurotrophic factor, which stimulates cell migration through activation of tropomyosin-related kinase B (TrkB; also called NTRK2). Knockdown of TrkB in human lung cancer cell lines significantly decreased their migratory and metastatic ability in vitro and in vivo. In an autochthonous lung adenocarcinoma model driven by activated oncogenic Kras and p53 loss, TrkB deficiency significantly reduced metastasis. Hypoxia-inducible factor-1 directly regulated TrkB expression, and, in turn, TrkB activated Akt signaling in metastatic lung cancer cells. Finally, TrkB expression was correlated with metastasis in patient samples, and TrkB was detected more often in tumors that did not have Kras or epidermal growth factor receptor mutations. These studies demonstrate that TrkB is an important therapeutic target in metastatic lung adenocarcinoma.

Keywords: NSCLC; TrkB/NTRK2.

Fig. 1.

BDNF stimulates migration of human lung adenocarcinoma cell lines. (A) Human lung adenocarcinoma cell lines were plated into transwell assays with the indicated medias. n = 3. *P < 0.01. (B) Serum-free (SFM) and lymph node fibroblast conditioned media (CM) were incubated on protein array membranes. n = 1. (C) A549 transwell migration assays with control CM (Control IgG IP), BDNF-depleted CM (BDNF IP), or BDNF-depleted CM supplemented with BDNF (BDNF IP + Rescue). n = 3. *P < 0.01. (D) Quantitative PCR (qPCR) analysis showing BDNF expression of the indicated sorted cell populations from wild-type mouse lungs. n = 3. *P < 0.01. (E) ELISA showing BDNF protein levels in wild-type mouse tissues (n = 4). *P < 0.01 indicates a significant difference between that tissue and the amount of BDNF in the stomach. All error bars represent mean ± SEM.

Fig. 2.

TrkB is required for human lung adenocarcinoma cell line migration and metastasis. (A) Transwell assays showing migration of the indicated cell lines expressing shTrkB toward conditioned media. n = 3. *P < 0.05. (B) Quantification of lung tumor burden of three to five mice per group i.v.-injected with H2030-BrM3 shGFP, shTrkB, or shTrkB-2 cells (n = 2–4). *P < 0.01. (C) Transwell assays showing migration of the indicated cell lines treated with ANA-12. n = 2–3. *P < 0.03. (D) Transwell assays showing migration of H23 cells overexpressing empty vector, wild-type TrkB (TrkB-OE), or kinase dead TrkB (TrkB-K571M). n = 2. *P < 0.02. (E) Representative pAkt and Akt immunoblots of the protein extracts from H2030-BrM3 shGFP and shTrkB cells treated with BDNF. n = 3. (F) Transwell assays showing migration of shGFP and shTrkB H2030-BrM3 cells expressing empty vector (EV) control or constitutively active Akt. n = 3. *P < 0.02. All error bars represent mean ± SEM.

Fig. 3.

TrkB is required for metastasis in a murine model of lung cancer. (A) Quantification of lung tumor burden of Kras;p53 (n = 14) and Kras;p53;TrkB (n = 18) mice. (B) Average number of lung tumors of the mice analyzed in A. (C) Number of mice analyzed in A with lymph node metastases. (D) The average number of metastases per mouse analyzed in A. *P < 0.01. All error bars represent mean ± SEM.

Fig. 4.

HIF-1α induces TrkB expression in human lung adenocarcinoma cell lines. (A) qPCR analysis of TRKB expression of the indicated cell lines cultured in hypoxia or normoxia. n = 3–4. *P < 0.04. (B) qPCR analysis of TRKB expression of H2030-BrM3 cells expressing the indicated hairpins. n = 3. *P < 0.01. (C) Transwell assays showing migration of the indicated cell lines cultured in normoxia or hypoxia toward media with DMSO or K252A. n = 3. *P < 0.03. (D) qPCR using VEGF negative or positive control or TrkB promoter primers and DNA purified from HIF-1α ChIP in A549 cells incubated in normoxia or hypoxia. n = 3. The enrichment relative to β-actin and the input is shown. *P < 0.01. All error bars represent mean ± SEM.

Fig. 5.

High TrkB expression is correlated with poor lung adenocarcinoma patient prognosis. (A) qPCR analysis showing TRKB expression in stage IA-IIIA tumors (n = 18) relative to stage IIIB-IV human lung adenocarcinoma tumors (n = 4). *P < 0.01. (B) Presence of TrkB expression in genotyped human lung adenocarcinomas. (C) Presence of distant metastases and smoking status in patients at the time of diagnosis or following treatment. All error bars represent mean ± SEM.