Pre-clinical evaluation of novel anti-metastatic targets

Background: Radiotherapy is used in the treatment of over 50% of cancer patients and bar surgery, is the most effective cancer intervention. However, in the clinic secondary malignancies have been observed following radiotherapy and in vitro increased cell migration and invasion have been seen following radiation. The Src/FAK signalling pathway is known to play an important role in the metastatic phenotype through its involvement in cell adhesion, migration and invasion and we have previously demonstrated that radiotherapy can activate this pathway along with the phosphoinositide 3-kinase (PI3K) pathway, also associated with tumour metastases and an aggressive phenotype. Using pharmacological inhibitors, we have investigated combination approaches to evaluate whether Src and PI3K targeting is beneficial in a radiotherapy context, especially focusing on metastatic phenotype. We wished to relate pathway activation to cellular phenotype and increase understanding of the metastatic cascade, the processes involved and the signalling pathways taking the lead.Method: Using thyroid carcinoma cell lines FTC133 and 8505c the effects of Src inhibition using AZD0530, FAK inhibition using FAKi and PI3K inhibition using GDC-0941 were studied. The effects of radiotherapy alone, and in combination with the above inhibitors, were also studied. In vitro MTT, apoptosis and clonogenic assays were used to assess cell proliferation and cell survival and scratch assays, cell adhesion and cell spreading assays were used to assess the effects of the drugs on metastatic characteristics. In vivo tumour growth, survival and ex vivo clonogenics were used to measure the effects of AZD0530 and GDC-0941. Western blotting, immunofluorescence and immunohistochemistry was used to observe the effects on pathway activation and protein localisation.Results: Src and FAK inhibition reduced metastatic characteristics of thyroid carcinoma cell lines in vitro such as cell spreading and migration. FAK inhibition showed a greater effect on cell survival by MTT, clonogenic and apoptosis. In the thyroid carcinoma cell lines radiotherapy enhanced the metastatic phenotype. This was seen by enhanced activation of the Src and PI3K pathways, increased migration and invasion in vitro and enhanced tumour metastasis in vivo. By combining Src inhibition with radiation a reduction in metastatic characteristics was observed and by combining PI3K inhibition with radiotherapy radiosensitivity could be improved. With the triple combination of Src and PI3K inhibition with radiotherapy a significant reduction in cell survival was demonstrated in vitro compared to radiation alone and either inhibitor combined with radiation, with a corresponding significant reduction in tumour growth being observed in vivo. With the combination of Src and PI3K inhibition significant reductions in metastatic characteristics were also observed both in vitro and in vivo seen by a reduction in cell migration and tumour metastasis. Finally combined inhibition of the Src and PI3K pathway reduced the radiation enhanced activation of several pathways in vivo including Src and PI3K.Conclusions: Together these results suggest that the Src and PI3K pathways play a role in radiation enhanced metastatic characteristics in thyroid carcinoma and through combined inhibition of the pathway the negative effects of radiation, enhanced migration and invasion, can be inhibited and the cells can be made more radiosensitive. Full characterisation of the pathways involved in radiation induced motility and radioresistance will provide further rationale for combination therapies and provide potential for application of these therapies in the clinic.