NTs, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3, -4/5, and -6 are critical to the development of the human nervous system. Moreover, NTs are important for survival and maintenance of neurons [25
]. NT receptors include both the low-affinity p75NTR as well as the high-affinity Trk family of receptors, which include TrkA, B, and C. NGF preferentially binds to TrkA, BDNF and NT-4/5 bind TrkB, and NT-3 interacts with TrkC [17
]. Following NT binding, Trks homodimerize and autophosphorylate specific tyrosine residues for receptor kinase activation as well as for creating docking sites for protein-protein interaction [7
]. Downstream signals from Trks are known to influence PI3 Kinase, Akt, PKC, Ras, and Erk. As such, Trk signaling can affect apoptosis, growth and differentiation pathways which are critical targets for cancer therapeutics. Our work presented here has identified transient radiation-induced Trk activation, and that Trk inhibition promotes radioprotection while TrkA stimulation enhances radiation-induced death in vascular endothelial cells.
Much of what is known about Trk comes from studies of neuroblastoma and medulloblastoma, where Trk expression levels are prognostic (Reviewed in [21
]). In normal neurons, all three Trks are expressed and low levels are detectable in astrocytes. However, data surrounding Trk in glioma are controversial. Trks have been detected in astrocytic gliomas but not oligodendrogliomas [26
] with higher levels of TrkA and B expression in lower grade lesions, suggesting that these Trks may be involved in tumorigenesis. While one group that identified TrkA in the glioblastoma cell lines, U251, U87, and U373, found that the Trk inhibitor, K252a, can inhibit NGF-stimulated proliferation [22
], others have shown that overexpression of TrkA in U251MG cell lines can elicit differentiation and growth inhibition [15
]. As such, the role(s) of Trk in glioma remains unclear.
More recently, investigations into the importance of Trk in non-CNS tissues have developed. Interestingly, Trk signaling has been implicated in Wilms Tumor, multiple myeloma, thyroid, lung, pancreas and prostate cancer [7
]. Moreover, evidence suggests that TrkB can promote neoangiogenesis [7
] while NGF stimulation of TrkA promotes angiogenesis in vivo
]. Taken together, Trk appears to be an important player in tumor development as well as response to therapy. Because we saw similar results with both the TrkA specific inhibitor and the pan-Trk inhibitor K252a, we suspect that TrkA may be the key family member in terms of radiation response. Our findings are particularly striking considering that Trk inhibitors, such as K252a, tend to promote cell death [19
] rather than promote a protective affect as we show here. However, it should be noted that the doses of K252a that have been used in such studies are 10-fold higher than the doses used in our work.
The direct evaluation of kinase activity described herein provides the proof of principle for using kinomic technology for time course and treatment response studies. Indeed, despite detecting only a transient Trk activation with irradiation, these studies demonstrate that Trk activity promotes radiation sensitization in vascular endothelial cells since Trk stimulation enhanced radiation effect while Trk inhibition radioprotected. As such, the Trk family may be a new class of molecular targets for modulating radiation effect in vasculature. The radioprotection that is promoted through TrkA inhibition and Trk family inhibition (K252a) appears to involve enhanced DNA DSB repair as we detected a faster rate of resolution of γH2AX foci in the irradiated samples. This enhanced repair utilizes the NHEJ pathway based on enhanced phospho-DNA-PKcs foci formation following radiation in the Trk inhibited cells. We were unable to detect significant differences in Rad-51 foci levels between control and treated cells suggesting that HR-mediated repair may not be essential in mediating this radioprotective response.
Although our studies were limited to radiation treatments, this kinomic profiling strategy can be applied to chemotherapeutics and molecularly targeted agents. Currently, prospective trials are investigating whether or not the genomic “signatures” of patient tumors can more accurately determine prognosis than standard clinicopathological features. Unfortunately, molecular biomarker utilization for predicting response to molecularly targeted agents has been fraught with problems. For example, EGFR expression levels have not correlated with EGFR kinase inhibition response consistently among disease sites [1
], do not necessarily equate with activation [11
], and identification of kinase mutations has further complicated studies for predicting response [1
]. Thus, a kinase activity-based assessment provides a more rational approach to the study of kinase inhibitors. Indeed, by using a kinase assay, resistance from any source, be it overexpression, mutation, or other “short-circuits” should be detectable. This information could not only provide prognostic and predictive information but also generate hypotheses regarding pathways for treatment resistance. The specific findings of this report suggest that further work is warranted to determine whether the manipulation of TrkA expression may lead to new cancer therapeutic strategies.