Abstract
Background/Aim: Tropomyosin-related kinase B (TrkB)/brain-derived neurotrophic factor (BDNF) signaling plays a role in inducing malignant phenotypes in several aggressive types of cancers. To create a conclusive therapy targeting TrkB/BDNF signaling in solid refractory cancers, the biological significance of TrkB/BDNF signaling was analyzed in pancreatic ductal adenocarcinoma (PDAC) cells. Materials and Methods: Three PDAC cell lines were used as target cells to investigate proliferation and invasiveness. Small interfering RNA (siRNA) and the TrkB tyrosine kinase inhibitor k252a were used as TrkB/BDNF signaling inhibitors. Results: All PDAC cell lines expressed TrkB and BDNF. When TrkB and BDNF were inhibited by siRNA or k252a, the invasiveness of PANC-1 and SUIT-2 cells significantly decreased. When TrkB was inhibited by siRNA or k252a, proliferation was significantly inhibited in PDAC cells. Conclusion: TrkB/BDNF signaling may be a new therapeutic target for PDAC. Therapies targeting TrkB/BDNF signaling may be a conclusive cancer therapy for refractory solid cancer.
Pancreatic ductal adenocarcinoma (PDAC) is a refractory cancer phenotype (1). Surgical resection is the only curative effective therapy; however, its indication is extremely low because the disease has typically already advanced at the time of diagnosis. Meanwhile, the types and effects of chemotherapeutic agents remain limited. Currently, there are no effective treatments for advanced and recurrent diseases. Therefore, the development of new therapeutic strategies is urgently required.
Tropomyosin-related kinase B (TrkB) is a member of the Trk family and acts as a receptor tyrosine kinase for brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor family. In normal cells, TrkB/BDNF signaling plays a role in the development of the nervous system (2, 3). However, in cancer cells, it has been shown that TrkB/BDNF signaling plays a role in inducing the aggressive phenotype of several cancers and is a factor of poor prognosis (4-6). TrkB/BDNF signaling is expected to be a promising therapeutic target for refractory solid cancers.
In this study, we investigated the biological significance of TrkB/BDNF signaling in PDAC to develop an effective therapy for PDAC.
Materials and Methods
Cell culture and reagents. Three human PDAC cell lines (ASPC-1, SUIT-2, and PANC-1) were maintained in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal calf serum (FCS; Life Technologies, Grand Island, NY, USA) and antibiotics (100 units/ml of penicillin and 100 μg/ml of streptomycin; Meijiseika, Tokyo, Japan).
Western blot analysis. Western blotting was performed as previously described (7). Protein samples (50 μg) were separated by electrophoresis on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to Protran nitrocellulose membranes (Whatman, Dassel, Germany). The protein-transferred membranes were incubated overnight at 4˚C with primary antibodies for TrkB (1:200, sc-8316, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and BDNF (1:200, sc-546, Santa Cruz Biotechnology). The membranes were then incubated with peroxidase-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) for 1 h at room temperature. The antibody for α-tubulin (1:1,000, Sigma-Aldrich, St. Louis, MO, USA) was used as a protein loading control.
RNA interference. ON-TARGETplus™ SMARTpool small interfering RNA (siRNA) targeting TrkB (L-003160), BDNF (L-017626), and negative control siRNA (ON-TARGETplus™ Control non-targeting siRNA, D-001810) were purchased from Dharmacon (Lafayette, CO, USA). Cells (0.2×106 cells/well) seeded in six-well plates were transfected with 100 nM siRNA using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were used for experiments at 2 days after transfection.
Invasion assay. Invasion assay was performed with a Matrigel-coated Transwell insert as previously described (8). Briefly, cells (2×105) were placed in the upper chamber and incubated for 18 h. The cells that migrated to the lower side of the filter were fixed and stained with Diff-Quik reagent (Sysmex, Kobe, Japan) and then counted under a light microscope (Nikon Eclipse TE 300, Nikon, Tokyo).
Cell proliferation assay. All PDAC cell lines were seeded into 96-well plates at 5,000 cells/well and cultured for 16 h. Cell proliferation was assessed by absorbance (Biotrak visible plate reader, Amersham Biosciences) at 492 nm (reference wavelength 620 nm) using Cell Count Reagent SF (Nacalai Tesque). In some experiments, recombinant BDNF (Peprotech, Rocky Hill, NJ, USA) and TrkB tyrosine kinase inhibitor: K252a (Alomone Labs, Jerusalem, Israel) were added to the culture.
Statistical analyses. The data are presented as the mean±standard deviation (SD). Calculations were carried out using JMP 12.0 software (SAS Institute, Cary, NC, USA) or Microsoft Excel software (Microsoft, Redmond, WA, USA). Student’s t-test was used to compare continuous variables between pairs of groups. A p- value <0.05 was considered significant.
Results
TrkB/BDNF signaling contributes to the invasiveness of PDAC cells. First, the expression of TrkB and BDNF in PDAC cells was confirmed by western blotting. All PDAC cell lines expressed TrkB and BDNF (Figure 1). Next, we investigated whether TrkB/BDNF signaling is involved in the invasiveness of PDAC cells. When TrkB and BDNF were inhibited by siRNA, the invasiveness of PANC-1 and SUIT-2 cells significantly decreased (Figure 2A). When TrkB/BDNF signaling was inhibited by k252a, which is an inhibitor of TrkB tyrosine kinase, the invasiveness of PANC-1 and SUIT-2 cells also significantly decreased (Figure 2B). These results suggest that TrkB/BDNF signaling may contribute to the invasiveness of PDAC cells. However, even if TrkB/BDNF signaling was stimulated by recombinant BDNF, there was no significant change in invasiveness (Figure 2C).
TrkB and BDNF are expressed in all three cell lines used in the experiments. The protein expression of TrkB and BDNF was examined by western blot analysis. TrkB: Tropomyosin-related kinase B; BDNF: brain-derived neurotrophic factor.
TrkB/BDNF signaling contributes to the invasiveness of PDAC cells. A: The invasive abilities of PDAC cells transfected with TrkB siRNA or BDNF siRNA were estimated using a Matrigel invasion assay. B: The invasive abilities of PDAC cells treated with k252a at the indicated concentrations were estimated using a Matrigel invasion assay. C: The invasive abilities of PDAC cells treated with recombinant BDNF at the indicated concentrations were estimated using a Matrigel invasion assay. In the invasion assay, all cells that had migrated from the upper to the lower side of the filter were counted. Data are presented as means±standard deviations. *Significantly different at p<0.05. TrkB: Tropomyosin-related kinase B; BDNF: brain-derived neurotrophic factor; PDAC: pancreatic ductal adenocarcinoma; siRNA: small interfering RNA.
TrkB/BDNF signaling contributes to the proliferation of PDAC cells. We investigated whether TrkB/BDNF signaling is involved in PDAC cell proliferation. When TrkB was inhibited by siRNA, proliferation of all three PDAC cells significantly decreased, while inhibition of BDNF by siRNA did not affect the proliferation of PDAC cells (Figure 3A). When TrkB/BDNF signaling was inhibited by k252a, the proliferation of PANC-1 cells also significantly decreased (Figure 3B). However, even if TrkB/BDNF signaling was stimulated by recombinant BDNF, there was no significant change in invasiveness (Figure 3C). These results suggest that TrkB/BDNF signaling may contribute to the proliferation of PDAC cells.
TrkB/BDNF signaling contributes to the proliferation of PDAC cells. A: Cell numbers at the indicated days among PDAC cells transfected with TrkB siRNA or BDNF siRNA were counted using light microscopy. B: Cell numbers among PDAC cells treated with k252a at the indicated concentrations were counted using light microscopy. C: Cell numbers among PDAC cells treated with recombinant BDNF at the indicated concentrations were counted using light microscopy. Data are presented as means±standard deviations. *Significantly different at p<0.05. TrkB: Tropomyosin-related kinase B; BDNF: brain-derived neurotrophic factor; PDAC: pancreatic ductal adenocarcinoma; siRNA: small interfering RNA.
Discussion
In our study, recombinant BDNF did not affect the proliferation and invasiveness of PDAC cells. We believe that PDAC cells secrete BDNF, and this autocrine BDNF may be sufficient to activate TrkB/BDNF signaling. Among all three PDAC cell lines, it was difficult to decrease the invasiveness of AsPC-1 cells. It is also expected, that antitumor agents are not effective in all patients with PDAC. It is important to understand the differences in AsPC-1 cells compared to the other two cell lines. For example, in the small cell lung cancer (SCLC) line, comprising SBC-5 cells, Hedgehog (Hh) signaling is inversely correlated with TrkB/BDNF signaling, and inhibition of Hh signaling leads to the activation of TrkB/BDNF signaling to induce malignant phenotypes (9). Some factors or signaling pathways may contribute to this discrepancy. For example, with respect to the cancer microenvironment, which has received much attention in current research, TrkB/BDNF signaling is suggested to correlate with hypoxia-inducible factor 1α, which is an important transcriptional factor under hypoxic conditions (10). Research should proceed in this direction.
Trk inhibitors are under investigation in clinical trials for TRK fusion-positive solid tumors. For example, a clinical trial on larotrectinib (LOXO-101), a selective pan-TRK inhibitor, is ongoing for TRK fusion-positive solid tumors, and promising results have been reported (11-13). ONO-7579, a second-generation pan-TRK inhibitor, is expected to be a potent and selective agent that is orally administered (14). Although a hopeful result for PDAC has not yet been obtained, the information obtained in the present study will help improve the effectiveness of Trk inhibitors for PDAC. We believe that the drawback is that the Trk inhibitor is not a selective TrkB inhibitor. Among the Trk family, some studies have shown that TrkA acts as a tumor suppressor in neuroblastoma and colon cancer (15) and that TrkC is a good prognostic factor for medulloblastoma and colon cancer (16, 17). The development of a selective TrkB inhibitor may be difficult because its molecular structure is similar to the proteins of the Trk family, which must be considered.
Figure 4 shows our previous findings on TrkB/BDNF signaling in SCLC (9), gallbladder cancer (18, 19), neuroblastoma (10), large cell neuroendocrine tumors (20, 21), and lung squamous cell carcinoma (22). Studies that target TrkB/BDNF signaling are ongoing in the context of other types of cancers, such as brain glioblastoma and oral squamous cell carcinoma (23, 24).
Schematic results of our studies regarding TrkB/BDNF signaling. Therapies targeting TrkB/BDNF signaling may be a conclusive cancer therapy against refractory solid cancer. TrkB: Tropomyosin-related kinase B; BDNF: brain-derived neurotrophic factor.
In the present study, we revealed the possibility that TrkB/BDNF signaling may be a new therapeutic target for PDAC. In conclusion, therapies targeting TrkB/BDNF signaling may be a conclusive cancer therapy for refractory solid cancer.
Acknowledgements
The Authors thank Ms. Emi Onishi for her skillful technical assistance. This study was supported by JSPS KAKENHI Grant Numbers JP 18K08620, JP 19K09124, JP 19K09047, and JP 20K09180.
Footnotes
Authors’ Contributions
Yasuhiro Oyama was involved in the analysis of all experiments. Shinjiro Nagao, Lin Na and Masayo Umebayashi were involved in the acquisition and analysis of data. Kosuke Yanai and Akio Yamasaki were involved in the gene transfection. Katsuya Nakamura, Shuntaro Nagai and Akiko Fujimura were involved in the interpretation of data. Kazunori Nakayama, Takashi Morisaki and Hideya Onishi were involved in the design of the work.
Conflicts of Interest
The Authors declare no financial or commercial conflicts of interest regarding this study.
- Received May 22, 2021.
- Revision received June 28, 2021.
- Accepted June 29, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
