Tropomyosin-related Kinase B Promotes Distant Metastasis Of Colorectal Cancer Through Anoikis Suppression

Colorectal cancer (CRC) is one of the most common malignancies. Evidence from several independent studies suggests that the survival rate of CRC patients is associated with the extent of tumor invasion and metastasis. Metastasis is a major factor that is often responsible for the failure of surgical therapy and chemotherapy. Increasing evidence shows that anoikis suppression plays an important role in distant metastases formation. The neurotrophic receptor tropomyosin-related kinase B (TrkB) is a receptor tyrosine kinase for which brain-derived neurotrophic factor is one of its primary ligands, and descriptions indicate its importance in anoikis suppression. Previous studies have demonstrated that TrkB is oncogenic in tumors and participates in cell proliferation, migration, invasion and anoikis detachment-induced apoptosis inhibition. Support for a role of TrkB in human tumors first came from studies on its expression pattern in neuroblastoma. Besides, TrkB is overexpressed by solid tumors of the lung, prostate pancreas and ovaries. In gastric cancer patients in particular, high TrkB expression is a independent prognostic marker. Thus, the significant overexpression of TrkB plays an important role in cancer. New approaches based on increased understanding of the biological and molecular nature of CRC are required. Understanding of the important mechanism of CRC distant metastatic capacity will allow the development of novel therapies that may promote anoikis.Objectives:Our aim was to analyze the expression and initial function of TrkB in vitro and in vivo, and to evaluate the effect of TrkB-induced anoisis supression in CRC. Our second aim was to detect the TrkB-regulated effective anoikis-related factor through anoikes assays.Our third aim was to detect the positive rate of TrkB expressions in CRC tissue through immunohistochemistry, and study the relationshiop with CRC patients survival.Methods:In this study, we investigated the TrkB expression levels and whether TrkB regulates anoikis suppression in CRC cells. Experssion vector of TrkB and shRNA-TrkB vector were constructed. The expression levels of TrkB in TrkB-SW480cells and shRNA-TrkB-LoVo cells were determined by Western Blot. Anoikis assays were performed flow cytometry analysis. An experimental metastasis assay was used to assess the distant metastatic properties in the shRNA-TrkB LoVo cells in vivo as compared to the control shRNA. Furthermore, we examined the critical signaling pathway of TrkB which promoting distant metastasis formation in CRC. The Akt activation was determined by Western Blot. Akt inhibitor was used to examine whether TrkB induced anoikis suppression through the Akt pathway. Finally, we analyzed the TrkB expression levels in surgical tissue samples through immunohistochemistry. And we assessed the relationship between TrkB expression and patient outcomes.Results:The expression levels of TrkB in CRC cells were determined by Western Blot, the results showed that the TrkB expressions of SW480, HCT116, HT29and LoVo were higher than that of GES. The TrkB expressions of SW480cells were significantly lower than that of the other CRC cells. And the TrkB expressions of LoVo cells were higher than that of the other CRC cells. We analyzed the TrkB expression levels in surgical tissue samples of60patients through immunohistochemistry, the positive expression rate in CRC tissue were significantly higher than the rate of positive expression in normal clonic mucosa tussue (P <0.001). We established a TrkB knockdown vector that expressed TrkB-specific shRNA and produced lentiviral particles for subsequent infection. The LoVo cells were infected with shRNA-TrkB lentiviral particle solution, and the control LoVo cells were infected with its empty vector. We established shRNA-TrkB LoVo cells that stably expressed shRNA-TrkB. We established TrkB-SW480cells that stably expressed TrkB. TrkB expression and Akt activation in the LoVo cells containing the shRNA-encoding recombinant vector were also assessed. The TrkB mRNA and protein expression levels were reduced. The shRNA-TrkB LoVo cells underwent massive apoptosis in suspension compared with the cells transduced with an empty vector. We carried out an experimental metastasis assay, the control LoVo cells had resulted in large secondary tumors in the lungs.In contrast to that of mice injected with shRNA-TrkB LoVo cells, significantly increasing multiple foci were apparent on the lungs of the nude mice injected with the control LoVo cells13to15days after injection. We found that Akt activation was effectively suppressed in the shRNA-TrkB LoVo cells as compared with the control LoVo cells, whereas Akt activation was maintained in the cells treated with control shRNA as compared with the untreated LoVo cells. Akt activation was significantly blocked in the LoVo cells treated with the Akt inhibitor, while there was no obvious change in the TrkB expression levels. Anoikis was induced by Akt inhibition in LoVo cells as compared with that in LoVo cells not treated with the Akt inhibitor. We determined that patients with strong and moderate TrkB staining intensities had a higher risk of death than patients with weak staining intensity did (log-rank test, P=0.004). Cox model analysis showed that TrkB expression was a significant risk factor associated with patient survival.Conclusion:In summary, our data show that TrkB regulates anoikis suppression in CRC cells throug the downstream target Akt, and that its expression correlates with the risk of death in CR patients. Ultimately, these evidences may prove to be an attractive target for the development of therapy aimed at inducing anoikis in colorectal cancer and an indicator of unfavorable clinical outcome.