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Keywords:

  • TrkB;
  • colon cancer;
  • metastasis;
  • proliferation;
  • apoptosis;
  • invasion

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Yu Y, Zhang S, Wang X, Yang Z, Ou G. Overexpression of TrkB promotes the progression of colon cancer. APMIS 2010; 118: 188–95.

Studies have confirmed that TrkB plays important roles in facilitating metastasis in various types of malignant tumors. In the present study, 30 cases of colon cancer and matched non-tumors were examined for the expression of TrkB by Western blot. The expression of TrkB was also examined in 90 colon tumor sections by immunohistochemical methods, and D2-40 staining was used to evaluate the correlation between TrkB expression and lymphatic vessel density. To investigate the effects of TrkB on the progression of colon cancer, siRNA specific for TrkB was transfected into LoVo cells, and proliferation, apoptosis and invasion of trasfected cells were examined using MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], flow cytometry and Transwell assays, respectively. Our results showed that TrkB was up-regulated in colon tumors compared with the non-tumorous counterparts, and the overexpression of TrkB was closely correlated with lymphatic vessel density (LVD) and metastasis. Inhibition of TrkB by siRNA increased the apoptotic rates of transfected cells, while the numbers of proliferative and invasive cells were decreased. In summary, our data suggest that overexpression of TrkB in colon cancer possibly plays roles in inhibiting apoptosis, promoting proliferation and invasion, facilitating tumor progression by lymphangiogenesis-associated metastasis.

Colon cancer is the third most common cancer worldwide, and the incidence of colon cancer is increasing. The prognosis of patients with colon cancer principally correlates with the proliferative, apoptotic and invasive potentials of tumor cells, which are regulated by some critical genes. Tropomysin-related kinase B (TrkB) is a member of the Trk family, and functions as a receptor tyrosine kinase, which is necessary for the normal development of the nervous system (1, 2). Recent studies have shown the suppression of anoikis and induction of metastasis by TrkB (3, 4). TrkB is up-regulated in various primary human tumors, including neuroblastoma (5) and hepatocellular carcinoma (6), especially in metastatic gastric (7) and pancreatic tumors (8). TrkB is also reported to have intimate correlations with the prognosis of cancer patients (9–11) or angiogenesis (12). Therefore, the overexpression of TrkB might play an important role in the progression of malignant tumors.

Studies have shown that enhanced TrkB signaling promotes cell survival (13), and when activated, TrkB leads to the activation of downstream signaling molecules like phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) (14, 15), which results in the differential regulation of tumorous processes as apoptosis and especially metastasis. Although point mutation was observed in colorectal cancer (16), whether TrkB positively participates in primary colon cancer has not yet been determined.

This study was designed to investigate the expression and clinical significance of TrkB in surgically resected colon cancer with different clinicopathological features. We report here that high TrkB expression is common in colon tumors, and increased expression of TrkB is correlated with LVD and lymph node metastasis. We also report here that the interruption of TrkB expression by TrkB-siRNA promoted apoptosis of LoVo cells. However, suppression of TrkB inhibited the proliferation and invasion of LoVo cells. These results identify TrkB as a potential promoter in the progression of colon cancer.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Tissue samples and patients

A total of 90 cases of colon tumors were collected from the Pathology Department of China Medical University. All the included patients had undergone surgical resection without any chemical or radiation therapy before. Formalin-fixed paraffin-embedded sections of tumor were stained with hematoxylin and eosin (H&E) routinely, and reviewed by two senior pathologists to determine the grade and stage of tumors, according to the TNM grading and staging system of colon tumors from AJCC (2002). Metastatic lymph nodes were determined by routine pathological examination of dissected nodes. Clinicopathological information of the patients regarding tumor size, differentiation, and stage, and lymph node metastasis is summarized in Table 1.

Table 1.   Clinicopathological characteristics of 90 colon adenocarcinomas and TrkB expression by immunohistochemistry
Clinicopathological characteristicsCases (n = 90)Higher TrkB expression (n = 65)Lower TrkB expression (n = 25)p-Value
  1. TrkB expression: *Significant difference between tumors with positive (+) and negative (−) lymph nodes; **significant difference between early (I + II) and advanced (III) stage of colon cancer.

Tumor size
 T < 5 cm3825130.407
 T ≥ 5 cm524012
Differentiation
 Well6043170.867
 Poor–moderate30228
Lymph node metastasis
 Without5132190.035*
 With39336
TNM stage
 I + II5132190.035**
 III39336

Cell culture and transfection

Human colon cancer LoVo cells with high expression of TrkB, as confirmed previously (data not shown), were preserved in our department and cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin, in an incubator with 5% CO2. To knock down the TrkB expression for subsequent experiments, LoVo cells (80–90% confluence) were transfected with TrkB-siRNA and the scrambled control (non-silencing) siRNA (both from Kaiji, Nanjing, China) for indicated time using Lipofectamin2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The experiments involving cells were repeated at least three times.

Western blot

Frozen tissues (including tumor and non-tumorous portion) or cells were washed twice with ice-cold phosphate buffered saline (PBS), homogenized on ice in lysis buffer containing 20 mM Tris–HCl, 1 mM EDTA, 50 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1% Triton X-100 and 1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenate was centrifuged at 22000 g for 30 min at 4 °C. The supernatant was collected and protein content was determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Eighty micrograms of total protein was separated by 6% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% bovine serum albumin (BSA), primary antibodies were incubated on the membranes for TrkB (sc-8316, 1:200) and β-actin (1:200) (both from Santa Cruz, CA, USA) overnight at 4 °C. The membranes were then incubated for 2 h at 37 °C with goat anti-rabbit IgG (1:2000) (ZhongShan, Beijing, China). Immunoreactive straps were identified using the enhanced chemiluminescence (ECL) system (Kaiji, Nanjing, China), according to the manufacturer’s instructions. The DNR Imaging System was used to catch up the specific bands, and the optical density of each band was measured using the Image J software. The ratio between the optical density of TrkB and β-actin of the same sample was calculated as relative content and expressed graphically.

Immunohistochemistry

Ninety paraffin sections of colon tumor were deparaffinized and rehydrated routinely. The antigen recovery was performed by heating slides in an autoclave for 2 min in 0.1 mol/l Tris–HCl buffer at pH 10. The sections were incubated overnight with primary rabbit polyclonal antibody detecting TrkB (sc-8316, 1:100) and mouse monoclonal antibody for D2-40 (ZhongShan), following 3% H2O2 and 5% rabbit serum treatment at 37 °C for 1 h. Then, they were incubated with secondary antibody and SP complex for 30 min (SP kit), and visualized with DAB (DAB kit) (both from MaiXin, Fuzhou, China). Negative controls were non-immune rabbit IgG at the same dilution as for the primary antibody. All sections were evaluated by two senior pathologists. Cells with brown particles in the membrane or cytoplasm were regarded as positive cells. The intensity of TrkB immunostaining (1 = weak, 2 = intense) and the percentage of positive tumor cells (0% = negative, 1–50% = 1, 51–75% = 2, ≥76% = 3) were evaluated in at least five high-power fields (×400 magnification). The scores of each sample were multiplied to give a final score of 0, 1, 2, 3, 4, or 6, and the tumors were finally determined as negative: score 0; lower expression: score ≤3; or higher expression: score > 3. Lymphatic vessels revealed by D2-40 immunostaining were counted in the peritumoral regions, and areas with more lymphatic vessels were chosen under a low-power (×100) field. LVD was evaluated as the mean value of vessel counts in randomly selected five low-power fields.

MTT assay

The MTT assay was performed to evaluate the proliferation of transfected cells. Cells were detached and seeded in five 96-well plates (1 × 103/200 μl) in parallel, and transfected for 24 h before detection. Everyday, 20 μl MTT (5 mg/ml) was added to each well of one plate, and 4 h later, the liquids were removed and 150 μl DMSO was added. After 10 min of vortexing, the optical density was measured. The cell growth curves were drawn according to time (X-axis) and average optical density (Y-axis). Data presented are representative of three individual wells.

Cell apoptosis assay

Cell apoptosis was examined by flow cytometry using an Annexin V-FITC (fluorescein isothiocyanate) apoptosis detection kit (BD Biosciences, San Jose, CA, USA), following the manufacturer’s protocol. Cells were washed twice in ice-cold PBS and resuspended in 1× binding buffer (1 × 106/ml). Cell suspension of 100 μl (1 × 105 cells) was gently mixed with 5 μl Annexin V-FITC and 5 μl PI, and then incubated for 15 min at room temperature away from light. After addition of further 400 μl of 1× binding buffer, cell apoptosis was detected in a flow cytometer. Results are representative of three individual experiments.

Cell invasion assay

The cell invasion assay was performed using a 24-well Transwell chamber (Costar, Cambridge, MA, USA). At 24 h following transfection, cells (1 × 104) were detached and seeded in the upper chamber of a 8 μm pore size insert pre-coated with Matrigel (BD) and cultured for another 24 h. Cells were allowed to migrate toward the medium containing 15% FBS in the bottom chamber. The non-migratory cells on the upper membrane surface were removed with a cotton tip, and the migratory cells attached to the lower membrane surface were fixed in 4% paraformaldehyde and stained with hematoxylin. The number of migrated cells was counted in five randomly selected 400× power fields under the microscope. Data presented are representative of three individual wells.

Statistical analysis

The SPSS 13.0 software was applied to complete data processing. ‘Nonparametric test’ was used to assess the levels of TrkB in colon tumors and non-tumorous counterparts. ‘Binary logistic regression’ analysis was applied to analyze the correlations between TrkB expression and clinicopathological characteristics, and ‘independent samples t-test’ was used to compare LVD between groups of higher and lower TrkB expression. One-way ANOVA was used to compare the differences between cells with various treatments. All data were represented as mean ± SD, and the results were considered statistically significant when the p-value was less than 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

TrkB expression by Western blot

Western blot analysis was used to evaluate TrkB expression in 30 colon tumors and non-tumorous tissues distant from the primary tumor of the same case. The overexpression of TrkB was found in 26 tumor samples in comparison with the non-tumorous counterparts (p = 0.000). The specific bands for TrkB of eight samples are shown in Fig. 1A, and the relative optical density of the tumorous (T) and non-tumorous (N) tissues of the same patient was measured and expressed graphically (Fig. 1B).

image

Figure 1.  (A) Expression of TrkB by Western blot in matched tumors (T) and non-tumors (N) from four of 30 colon cancer patients. Significant TrkB overexpression was observed in tumorous in comparison with the non-tumorous tissue of the same case. (B) The ratio between the optical density of TrkB and β-actin of the same sample was calculated. The significant difference of TrkB expression between tumors (T) and non-tumors (N) was analyzed statistically and expressed graphically. TrkB immunoreactivity was greater in neoplastic tissues (p = 0.000).

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TrkB expression in 90 cases of colon cancer by immunohistochemistry

TrkB immunoreactivity was detected in 74 (82.2%) colon tumors. We considered that 65 tumors (72.2%) were of higher expression (scores of 4 or 6) and 25 cases (27.8%) were of lower expression (scores of 0, 1, 2, or 3), as described in Materials and methods. Clinicopathological correlations of TrkB expression revealed by immunohistochemistry were then analyzed statistically. No statistical differences were found between the higher TrkB expression and the characteristics of tumor size (T ≥ 5cm vs T < 5cm, p = 0.407) as well as differentiation (well vs poor-moderate, p = 0.867). However, immunostaining showed a statistically significant correlation between higher TrkB expression and lymph node metastasis at the time of resection (p = 0.035). Higher TrkB expression and advanced stage of colon cancer (I + II vs III, p = 0.035) were also observed in parallel. TrkB has been reported to induce tumor metastasis possibly by angiogenesis (17) and we wondered here whether the overexpression of TrkB in colon cancer was correlated with lymphoangiogenesis, which regularly facilitates tumor metastasis (18). D2-40 has been used as a biomarker for lymphatic vessels (19, 20) and was used in this study for LVD analysis, accordingly. Our results showed that LVD in positive and negative node groups were 169.0 ± 29.1 and 125.7 ± 37.5, respectively. More lymphatic vessels were present in tumors with higher TrkB expression (p = 0.000). Samples of TrkB expression in colon tumors with or without lymph node metastasis and the lymphatic vessels represented by D2-40 immunostaining under the same microscopic field are shown in Fig. 2. The correlations between TrkB expression and clinicopathological characteristics are shown in Table 1.

image

Figure 2.  TrkB expression and LVD evaluation by immunohistochemistry. (A) Intense immunoreactivity of TrkB was found in colon cancer with lymph node metastasis. (B) More lymphatic vessels revealed by D2-40 immunostaining were shown under the same microscopic field. (C) Poor expression of TrkB was observed in colon cancer without lymph node metastasis. (D) A small number of lymphatic vessels were found in the same case. It was shown that the high expression of TrkB was positively correlated with lymph node metastasis (p = 0.041) and LVD (p = 0.000) in colon cancer. Original magnification: all ×200.

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Inhibition of cell proliferation by TrkB-siRNA

LoVo cells with higher expression of TrkB were used in this study to investigate the effects of specific siRNA for TrkB on proliferation, apoptosis, and invasion of transfected cells. Figure 3A shows the inhibition of TrkB in transfected cells. We found that 5 days after TrkB-siRNA transfection, the O.D. of TrkB-siRNA, non-silencing siRNA and control groups were 1.41 ± 0.09, 1.58 ± 0.02, and 1.55 ± 0.06, respectively (p = 0.035). From the proliferative curves, we observed that the cells of the TrkB-siRNA group showed inhibited proliferation during the 5 days examined compared with the non-silencing and control groups (Fig. 3B).

image

Figure 3.  Inhibition of cell proliferation by TrkB-siRNA. (A) TrkB-siRNA-transfected LoVo cells exhibited decreased TrkB expression in comparison with non-silencing siRNA and control groups. (B) The growth curves showed the less tendency to proliferation of TrkB-siRNA treated cells during the 5 days examined compared with non-silencing and control group. The data are represented as mean ± SD of three independent experiments.

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Increased apoptotic rate in TrkB-siRNA-transfected cells

A negative role of TrkB in apoptosis of various tumor cells has been confirmed recently (21, 22). Therefore, apoptosis was detected after TrkB-siRNA transfection using Annexin V-FITC assay by flow cytometry. The decreased expression of TrkB was also confirmed in TrkB-siRNA transfected cells (Fig. 4A). The apoptotic rates of LoVo cells in TrkB-siRNA, non-silencing and control group were 26.5 ± 1.9%, 8.1 ± 0.5% and 5.4 ± 1.2%, respectively (p = 0.000, Fig. 4B).

image

Figure 4.  TrkB-siRNA promoted cell apoptosis. (A) TrkB-siRNA-transfected LoVo cells showed suppressed expression of TrkB. (B) The apoptotic rate of cells with TrkB knockdown was apparently increased in contrast to non-silencing siRNA and control groups. The results are indicated as mean ± SD of three individual tests.

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Effect of TrkB-siRNA on the invasive potential of transfected cells

Reports have shown that TrkB overexpression was found in more aggressive tumors (23). We next transiently inhibited TrkB by specific siRNA to determine the contribution of TrkB on invasion of LoVo cells. As shown in Fig. 5A, TrkB-siRNA-transfected cells exhibited extremely diminished TrkB in comparison with non-silencing siRNA and control groups. The numbers of invasive cells in TrkB-siRNA, non-silencing and control group were 16.2 ± 3.9, 26.4 ± 3.2 and 25.9 ± 3.3, respectively (p = 0.020, Fig. 5B).

image

Figure 5.  Interruption of cell invasion by TrkB-siRNA. (A) Specific siRNA inhibited TrkB expression in LoVo cells. (B) The number of invasive cells in TrkB-siRNA group was significantly reduced compared with that in non-silencing and control group. The values are mean ± SD of three replicates.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

TrkB is a receptor tyrosine kinase and mediates various signaling pathways. TrkB is critical to tumorigenesis (25) and metastasis (26), by promoting cell proliferation and survival. TrkB expression was found up-regulated in a variety of human tumors including neuroblastoma, ovarian cancer, and pancreatic tumors. Targeting at interfering TrkB expression may be helpful in the progression of effective anticancer therapies.

This study investigated TrkB expression to determine the clinical significance of TrkB in the progression of colon cancer. We examined 30 colon tumors and paired non-tumors by Western blot, and found a statistically significant TrkB overexpression in colon cancer. TrkB expression was significantly higher in neoplastic tissues, which suggested that TrkB overexpression correlates with the proliferation and survival of colon tumor cells. Despite the need for further research, these data suggest a crucial role of TrkB in facilitating tumorigenesis of colon cancer.

The expression of TrkB was also examined in 90 sections of colon cancer by means of immunohistochemistry. Through immunostaining, TrkB was detected extensively in colon tumors, further demonstrating that the overexpression of TrkB was common in colon tumors, regardless of tumor size and differentiation. A previous study has shown that TrkB expression was correlated with angiogenesis of ovarian cancer (12), while we found in this study that patients with higher TrkB expression had a higher LVD and a significant metastatic phenotype. These results indicate that TrkB probably facilitated metastasis of colon cancer partially by associated lymphoangiogenesis.

A study has shown that trk tyrosine kinase inhibitor induced cell death (27), and here we investigated the effects of TrkB-siRNA on the proliferation, apoptosis, and invasion of LoVo cells. After TrkB-siRNA transfection, LoVo cells exhibited a much lower level of TrkB and suppressed proliferation, which suggested that TrkB possibly promoted the proliferation and was likely necessary for the growth of LoVo cells. Studies have shown that TrkB inhibited anoikis of tumors (28), and we noticed that the apoptotic rate of transfected cells was increased by TrkB-siRNA, confirming the negative role of TrkB in cell apoptosis, which might help survival of normal LoVo cells. It is well accepted that the invasive ability of tumor cells plays a critical role for a successful metastasis. We observed in this study that the invasion of LoVo cells was largely inhibited by the deficiency of TrkB, indicating the important role of TrkB in LoVo cell invasion and even metastasis of colon cancer.

In conclusion, our study demonstrated that the overexpression of TrkB was common in colon cancer, which suggested that TrkB might play a critical role in inducing tumorigenesis of colon cancer. We also found that the increased expression of TrkB was correlated with higher metastatic ability, possibly by lymphoangiogenesis. The augmented apoptosis, attenuated proliferation and invasion of LoVo cells were also observed by the interruption of TrkB expression using specific siRNA. Taken together, TrkB may provide a helpful target for inhibitory therapies of progression of colon cancer. The regulatory signaling downstream of TrkB in colon cancer deserves further investigation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We are grateful to Chengyao Xie for technical assistance and Fanxin Meng for experimental instructions, and the staff of our department for useful suggestions.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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