Brain-Derived Neurotrophin and TrkB in Head and Neck Squamous Cell Carcinoma

Abstract

We hypothesized that in head and neck squamous cell carcinoma (HNSCC), the neurotrophin brain-derived neurotrophic factor (BDNF) and its high affinity receptor TrkB regulate tumor cell survival, invasion, and therapy resistance. We used in situ hybridization for BDNF and immunohistochemistry (IHC) for TrkB in 131 HNSCC samples. Brain-derived neurotrophic factor was highly expressed in normal mucosa in HNSCC tissue and in cell lines, whereas only 42.74% of HNSCC tissue was TrkB⁺. One fourth of HNSCC cases was human papilloma virus (HPV)^- positive, but the TrkB IHC frequency was not different in HPV-positive (HPV⁺) and negative cases. The UPCI-SCC090 cells expressed constitutive levels of TrkB. Transforming-growth-factor-β1 (1 ng/mL TGF-β1) induced TrkB in a subpopulation of SCC-25 cells. A single 10-µg/mL mitomycin C treatment in UPCI-SCC090 cells induced apoptosis and BDNF did not rescue them. The SCC-25 cells were resistant to the MMC treatment, and their growth decreased after TGF-β1 treatment, but was restored by BDNF if it followed TGF-β1. Taken together, BDNF might be ineffective in HPV⁺ HNSCC patients. In HPV^- HNSCC patients, tumor cells did not die after chemotherapeutic challenge and BDNF with TGF-β1 could improve tumor cell survival and contribute to worse patient prognosis.

Keywords: cytokeratin; epithelial–mesenchymal transition; p53 mutation; paraffin embedding; riboprobe; vimentin.

Figure 1

mRNA expression of brain-derived neurotrophic factor (BDNF) and NTRK2, protein synthesis of NTRK2 in head and neck squamous cell carcinoma (HNSCC) (A,B): In situ hybridization of antisense (A) and sense (B) riboprobe for BDNF (blue) in larynx SCC, cell nuclei counterstained in nuclear fast red. The antisense probe shows intensive purple—blue reactive areas, while the tissue reacted with the sense probe is slightly purple—blue stained. (C): In situ hybridization of antisense BDNF riboprobe and (D): immunohistochemistry of TrkB (brown) in tumor cell nests of oral SCC. A and B and C and D are sequential sections. (E): PCR detection of BDNF (519 base pairs, bps), NTRK2 (full protein coding area, 2528 bps) normalized to loading control ACTB (534 bps, not shown, normalized values represented as column bars) gene expression in cDNA samples of control UPPP normal mucosa, immunohistochemically (IHC) TrkB-positive and TrkB-negative HNSCC. BDNF is expressed in both normal and malignant tissue, NTRK2 is not present in normal mucosa, but if positive TrkB IHC staining was detected, the NTRK2 gene expression was also confirmed by PCR, while TrkB-negative IHC was also negative in RT-PCR. (A–D) images were taken by the TissueFaxs system, bars: 200 µm: (A,B); 100 µm: (C,D). Bands densitometry was done using Azurespot 14.2.

Figure 2

Relationship of TrkB immunostaining with N-stage, recurrence or second tumor and with p53 immunohistochemistry. TrkB was detected in 56 of 131 HNSCC cases and was scored semi-quantitatively as described in the Materials and Methods section. The semiquantitative values did not show normal distribution as tested by D’Agostino and Pearson normality test. The IHC score values were displayed as dot plots of mean and standard error of measurement (SEM). (A) N-stage data was available in 96 cases with the following case numbers: N0: 25, N1: 13, N2: 54 and N3: 4. N1–N3 showing increasing severity of lymph node metastasis also showed increase tendency in the mean value of the frequency of TrkB⁺ cases, but this difference was not statistically significant using Dunn’s multiple comparisons test. (B) Comparing primary (n = 97) and secondary or recurrent HNSCC (n = 23) the TrkB staining was significantly more frequent in secondary or recurrent HNSCC than in primary tumor. Statistical comparison was performed using Mann–Whitney test. (C) Comparing HNSCC with normal (wild-type; n = 54) TP53 and with altered (n = 76) TP53 immunohistochemistry, the TrkB staining was significantly more frequent in cases with altered TP53 than in cases with normal TP53. Statistical comparison was performed using Mann–Whitney test.

Figure 3

Gene expression of BDNF and NTRK2 in SCC-25 and UPCI-SCC090 cells, and immunohistochemical reaction of TrkB in paraffin embedded HNSCC cell lines. (A) Using an optimized primer designed for a 519 bp PCR product of BDNF and for a 620 bp PCR product of NTRK2 gene expression, both were detected in both SCC-25 and UPCI-SCC090 HNSCC cell lines. BDNF and NTRK2 levels were higher in UPCI-SCC090 cells. The column bars represent mean ± SEM. Statistical comparison is not available due to the relative low number of measurements. ACTB was used as loading control and its optical density values were used for normalization of both BDNF and NTRK2 optical densities. (B) If whole protein coding region of corresponding 2528 base pairs of NTRK2 was amplified, only in UPCI-SCC090 cells was a PCR product available. At protein level using paraffin embedded cells, TrkB was not present in Detroit 562 (C), and was only in few scattered cells in SCC-25 ((D), arrow) cell line, but was present in all cells in UPCI-SCC090 (E) cell line. TrkB was stained using the rabbit polyclonal 80G2 antibody and was detected in brown. Cell nuclei were counterstained in blue by hematoxylin. Bars: 100 µm.

Figure 4

Apoptosis detection in paraffin-embedded UPCI-SCC090 cells by immunohistochemical reaction of cleaved caspase-3 in control and 25 ng/mL BDNF-treated conditions UPCI-SCC090 cells were counted, paraffin embedded, sectioned, and stained with cleaved caspase-3 specific rabbit monoclonal antibody 96 h after a single MMC treatment in control and 25 ng/mL BDNF-treated conditions. The antibody reaction was detected in brown, cell nuclei were counterstained with hematoxylin in blue. Both control and BDNF-treated cells show a significant portion of dark brown cleaved-caspase-3-positive apoptotic cells (blue arrows). Bars: 20 µm. Images were taken with the TissueFaxs ^® system. The white boxes in the left corner of the images represent the whole section, the red part in these white boxes shows the position of the imaged area within the section.

Figure 5

Influence of BDNF on cell survival of UPCI-SCC090 and SCC-25 cells after MMC treatment UPCI-SCC090 and SCC-25 cells were plated in 6-well plates in serum-free medium or in medium supplemented with 1 ng/mL TGF-β1 (for SCC-25) or 25 ng/mL BDNF (for both) and cultured for 72 h followed by 30 min 10 µg/mL MMC treatment and 2-times 48 h treatment with TGF-β1 (for SCC-25) or BDNF. After the treatments, the cell numbers were counted and related to the original plated numbers ((A) UPCI-SCC090, (B) SCC25). One-thousand cells from each sample, represented on panels (A,B), were plated in 75-cm² cell culture flasks grown in serum-supplemented medium for 3 weeks and the growing colonies were stained with gentian violet and were counted. The number of growing colonies were related to plated cell numbers (1000). Treatment of BDNF or TGF-β1 did not cause any significant difference in the MMC surviving cell numbers of UPCI-SCC090 cells (A) or SCC-25 (B), BDNF treatment showed a non-significant increase in the cell numbers of both cell lines. The cell numbers data were normal distributed in UPCI-SCC090 cells, unpaired t-test did not show significant difference for BDNF-treatment. In SCC-25 cells the cell numbers data were not normally distributed, and Dunn’s multiple comparisons test did not show any significant difference for the treatments against the control. In case of SCC-25 cells, after three weeks, cell clones were growing in control and treated settings (C,D), the number of growing clones (stained in blue by gentian violet on D) was significantly lower in case of TGF-β1 treatment than in all other settings (C), but in combination of TGF-β1 treatment before MMC and BDNF after MMC the number of clones were significantly higher than in case of TGF-β1 treatment both before and after MMC application. UPCI-SCC090 cells did not form any clones three weeks after MMC treatment (E) either in control or in BDNF-treated settings. The number of growing clones related to plated cell numbers did not show normal distribution. Significance was claimed if the p-value was lower than 0.05 using Tukey’s multiple comparisons test.

Figure 6

BDNF-treatment induces EMT in UPCI-SCC090 cells. UPCI-SCC090 cells were plated in serum-containing medium, treated with 10 µg/mL MMC for 30 min followed by 25 ng/mL BDNF-treatment for 2 × 2 days. After completion of this procedure, cells were collected, embedded in agarose and in paraffin, 5 µm sections were cut, and these were stained with mouse monoclonal anti-pan-cytokeratin antibody (Ventana, detected in green) combined with rabbit monoclonal vimentin (SP-20) antibody (detected in red), and 4′,6-Diamidin-2-phenylindol (DAPI) (blue) cell nuclear counterstaining. The immunohistochemical reactions are shown combined (A,D) and separated (B,C,E,F) in control (A–C) and BDNF-treated (D–F) cells. The camera profile, fluorescence excitation light exposition conditions were the same by all images, and experiment profile was re-used. The BDNF-treatment lead to increased vimentin representation without the loss of cytokeratin signal. The mean intensity for the cytokeratin (detected by the 44Fl Zeiss Filter Channel) and vimentin (detected by the A594 Zeiss Filter Channel) signals in all cells have been plotted on color-mapped heat intensity diagrams in control (G) and in BDNF-treated conditions (H). These graphs also show an increase in the double-positive (cytokeratin–vimentin) cell population (G,H: red arrows; C,F: white arrows). Bars: 50 µm.

Figure 7

TGF-β1 regulates the protein levels of TrkB, together with epidermal growth factor receptor (EGFR) and TrkA in SCC-25 cells. (A) 2 × 10⁵ SCC-25 cells/10-cm dish were plated for three days. Then an adaption from serum to albumin medium followed over two days. After that the cells were treated with albumin medium supplemented with TGF-β1 (1 ng/mL) for 24 h. The next day the cells were used for protein extraction in Cell Lysis Buffer (Cell Signaling Technology). The concentration of the samples was measured, and they were diluted to the same concentration of 1 µg/µL. After that the cell lysates were processed on a PathScan^® RTK Signaling Antibody Array Kit (Cell Signaling Technology^®, #7949) according to the manufacturer’s instructions. A1–A2: positive control; B1–B2: EGFR (Her1); A3–A4: TrkA; B3–B4: TrkB. (B) 5 × 10⁴ SCC-25 cells/mL were plated in 75-cm² culture dishes (Orange Scientific, Braine-l’Alleud, Belgium,) in serum-free, albumin containing medium [20] and cultured for 72 h. After that, the cells were washed with PBS and incubated with 10 µg/mL Mitomycin C (Sigma–Aldrich^®, St. Louis, MI, USA) in serum-free albumin-containing medium for 30 min at 37 °C ensuring cell cycle arrest. Then the cells were washed twice with albumin-containing medium and subsequently treated with albumin-containing medium for two times 48 h supplied with 1 ng/mL recombinant human TGF-β1, or in control conditions without TGF-β1 for 96 h. After completion of treatments the cells were embedded in agarose and in paraffin as described before [28]. TrkB was stained using the rabbit polyclonal 80G2 antibody and was detected in brown. Cell nuclei were counterstained in blue by hematoxylin. Arrows represent positive cells. Bars: 100 µm.