Aim 1. To investigate biologic consequences of BDNF activation of TrkB on chemoresistance. A. AKT pathway inactivates Glycogen synthetase 3beta (GSK3) Previously, we identified the PI-3 kinase pathway as mediating BDNF/TrkB-induced resistance to etoposide, vincristine and adriamycin. Last year we identified that AKT played a key role in regulating chemoresistance in NB cells and identified inhibitors of the AKT that could enhance the efficacy of chemotherapy. Now we have identified that AKT suppresses GSK3B activity and the inactivation of GSK3 attenuates the effects of chemotherapy by altering the intrinsic apoptotic pathway. Moreover we find that if we can enhance the activity of GSK3, the neuroblastoma tumor cells are more sensitive to chemotherapy. Many pharmacologic anti-deppresants such as LiCl inhibit GSK3 activity and we found that these can make the tumor cells less resistant to chemotherapy. This raises the possibility that some types of anti-depressants may affect the efficacy of chemotherapy. B. BDNF activation of TrkB via MAPK pathway decreases pro-apoptotic BH-3 protein BIM, which contributes to paclitaxel resistance. The ability of a tumor cell to survive an apoptoic insult or survive in a new microenvironment depends on both the external stress (chemotherapy, nutrient and O2 levels), the microenvironment (e.g. metastasize) and the cells' internal signaling systems. Our study on Bim indicates that, depending on the chemotherapeutic drugs utilized, BDNF/TrkB activation attenuates the effects of cytotoxic agents by distinct signaling pathways. We used a candidate gene approach to investigate targets downstream of AKT involved in chemoresistance. Activated Akt phosphorylates and inhibits several pro-apoptotic proteins, such as Bad, caspase-9 and Forkhead transcription factors (FKHRL1, FKHR and AFX), leading to cell survival. We did not detect significant changes in phosphorylation or total protein levels for BAD or caspase 9 after BDNF activation of TrkB. In our AKT study, the genetic and pharmacologic inhibiton of AKT activity focused our attention on FKHRL1. FKHRL1 functions in apoptotic processes by regulating p27 interaction with caspase 8 and with the pro-apoptotic BH-3 protein BIM. As the NB cell lines under study did not express caspase 8, we focused on BIM. BIM is a target of AKT in many other systems. We found that BDNF/TrkB activation leads to a decrease in BIM levels that is mediated by activation of MAPK and not AKT. SiRNA mediated BIM knockdown had no effect on cisplatin/etoposide induced cytotoxicity. However, silencing of BIM or inhibition of the MAPK pathway sensitized NB cells to paclitaxel. Bim is thought to induce cell death by binding to LC8 cytoplasmic dynein light chain where it is sequestered with the microtubule-associated dynein motor complex in healthy cells. Certain apoptotic stimuli such as paclitaxel stabilize microtubules and disrupt the interaction between LC8 and the dynein motor complex, freeing Bim to translocate together with LC8 to Bcl-2 and neutralize its anti-apoptotic activity. In our study, reduction of Bim by Bim siRNA, decreases paclitaxel induced apoptosis. It is becoming increasingly apparent that different drugs or toxic insults utilize distinct apoptotic signaling paths just as survival signaling pathways use distinct pathways to attenuate death signals. Clinically, only 8/33 NB patients using an """"""""up-front phase II window"""""""" approach had objective responses with paclitaxel which limited its potential utility when combined with current multi-agent regimens. Our study would indicate that drugs previously thought to have marginal activity may need to be re-evaluated in combination studies when appropriate signal transduction inhibitors become available.
Specific Aim 2. To investigate biologic consequences of BDNF activation of TrkB on metastasis and angiogenesis A. In a collaborative project we have found that BDNF activation of TrkB can stimulate expression of chemokines in the brain. This has enormous implications as chemokines have been implicated in cell movement which is required for metastasis of tumor cells. This finding contributes to our understanding of how BDNF activation of TrkB stimulates tumor cell movement.
| Ahmed, Farid; Tessarollo, Lino; Thiele, Carol et al. (2008) Brain-derived neurotrophic factor modulates expression of chemokine receptors in the brain. Brain Res 1227:1-11 |
| Li, Z; Zhang, J; Liu, Z et al. (2007) Downregulation of Bim by brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from paclitaxel but not etoposide or cisplatin-induced cell death. Cell Death Differ 14:318-26 |
