Ruth Ho

Trk Receptor Expression and Inhibition in Neuroblastomas

Neuroblastoma, the most common and deadly solid tumor in children, exhibits heterogeneous clinical behavior, from spontaneous regression to relentless progression. Current evidence suggests that the TRK family of neurotrophin receptors plays a critical role in these diverse behaviors. Neuroblastomas expressing TrkA are biologically favorable and prone to spontaneous regression or differentiation, depending on the absence or presence of its ligand (NGF) in the microenvironment. In contrast, TrkB-expressing tumors frequently have MYCN amplification and are very aggressive and often fatal tumors. These tumors also express the TrkB ligand (BDNF), resulting in an autocrine or paracrine survival pathway. Exposure to BDNF promotes survival, drug resistance, and angiogenesis of TrkB-expressing tumors. Here we review the role of Trks in normal development, the different functions of Trk isoforms, and the major Trk signaling pathways. We also review the roles these receptors play in the heterogeneous biological and clinical behavior of neuroblastomas, and the activation of Trk receptors in other cancers. Finally we address the progress that has been made in developing targeted therapy with Trk-selective inhibitors to treat neuroblastomas and other tumors with activated Trk expression.

Proliferation of Human Neuroblastomas Mediated by the Epidermal Growth Factor Receptor

Neuroblastoma is a common solid tumor of childhood that is derived from the neural crest. Expression of epidermal growth factor (EGF) receptors (EGFRs) has been associated with enhanced cell growth and aggressive behavior in other tumors. Here, we examined the expression profile of EGFRs in neuroblastoma cell lines and primary tumors. We found that all 13 neuroblastoma cell lines examined expressed EGFR1 (HER1), most at readily detectable levels. Low levels of other human EGFR family receptors were also detected in almost all cell lines. All primary tumors examined expressed readily detectable levels of HER1 and HER3 and lower levels of HER2 and HER4. EGF had a significant effect on the proliferation of neuroblastoma cell lines in vitro. EGF treatment (100 ng/mL) of the cell lines SY5Y and NLF significantly increased cell number (P < 0.01). EGF stimulated more cells to enter S and G2-M phase, as suggested by flow cytometry, indicating that EGF increases cell number by increasing proliferation, with no appreciable change in apoptosis. EGF exposure resulted in receptor autophosphorylation and activation of both the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. Exposure to 0.5 micromol/L ZD1839, a HER1-specific inhibitor, caused a 40% to 50% reduction in the number of SY5Y and NLF cells grown in medium containing 10% fetal bovine serum (P < 0.01). Even at 0.01 micromol/L, ZD1839 inhibited autophosphorylation of HER1 by EGF. At 0.1 micromol/L, it also blocked phosphorylation of AKT, but not MAPK, in NLF cells. Additional studies showed that the PI3K/AKT-specific inhibitor LY294002 had a more profound effect than the MAPK-specific inhibitor U0126 in blocking EGF-induced cell proliferation. This suggests that the PI3K/AKT pathway is the main signaling pathway responsible for the proliferation effects of EGF in neuroblastomas. Our results also indicate that ZD1839 is a potent inhibitor of neuroblastoma cell proliferation; therefore, it may be a useful, biologically based therapeutic agent for these tumors.

Lestaurtinib Enhances the Antitumor Efficacy of Chemotherapy in Murine Xenograft Models of Neuroblastoma

Purpose: Neuroblastoma, a common pediatric tumor of the sympathetic nervous system, is characterized by clinical heterogeneity. The Trk family neurotrophin receptors play an important role in this behavior. Expression of TrkA is associated with favorable clinical features and outcome, whereas TrkB expression is associated with an unfavorable prognosis. We wanted to determine if the Trk-selective inhibitor lestaurtinib had therapeutic efficacy in a preclinical neuroblastoma model. Experimental Design: We performed intervention trials of lestaurtinib alone or in combination with other agents in TrkB-overexpressing neuroblastoma xenograft models. Results: Lestaurtinib alone significantly inhibited tumor growth compared to vehicle-treated animals [P = 0.0004 for tumor size and P = 0.011 for event-free survival (EFS)]. Lestaurtinib also enhanced the antitumor efficacy of the combinations of topotecan plus cyclophosphamide (P < 0.0001 for size and P < 0.0001 for EFS) or irinotecan plus temozolomide (P = 0.011 for size and P = 0.012 for EFS). There was no additive benefit of combining either 13-cis-retinoic acid or fenretinide with lestaurtinib compared to lestaurtinib alone. There was dramatic growth inhibition combining lestaurtinib with bevacizumab (P < 0.0001), but this combination had substantial systemic toxicity. Conclusions: We show that lestaurtinib can inhibit the growth of neuroblastoma both in vitro and in vivo and can substantially enhance the efficacy of conventional chemotherapy, presumably by inhibition of the Trk/brain-derived neurotrophic factor autocrine survival pathway. It may also enhance the efficacy of selected biological agents, but further testing is required to rule out unanticipated toxicities. Our data support the incorporation of Trk inhibitors, such as lestaurtinib, in clinical trials of neuroblastoma or other tumors relying on Trk signaling pathways for survival. Clin Cancer Res; 16(5); 1478–85

Clinical Significance of NTRK Family Gene Expression in Neuroblastomas

Neuroblastomas (NBs) are characterized by clinical heterogeneity, from spontaneous regression to relentless progression. The pattern of NTRK family gene expression contributes to these disparate behaviors. TrkA/NTRK1 is expressed in favorable NBs that regress or differentiate, whereas TrkB/NTRK2 and its ligand brain‐derived neurotrophic factor (BDNF) are co‐expressed in unfavorable NBs, representing an autocrine survival pathway. We determined the significance of NTRK family gene expression in a large, representative set of primary NBs.

The effect of P75 on Trk receptors in neuroblastomas

Neuroblastomas (NBs) with favorable outcome usually express TrkA, whereas unfavorable NBs frequently express TrkB and its cognate ligand BDNF. P75 (p75(LNTR), NGFR, TNFRSF16) binds NGF-related neurotrophins with low affinity and usually is coexpressed with Trk receptors in NBs. Here, we investigated the importance of p75 coexpression with Trk receptors in NBs. We transfected p75 into two Trk-null NB cell lines, SH-SY5Y and NLF that were also engineered to stably express TrkA or TrkB. Cell numbers were compared between single (Trk alone) and double (Trk+p75) transfectants, and proliferation was assessed by flow cytometry. P75 coexpression had little effect on cell growth in Trk NB cells in the absence of ligand, but it increased sensitivity and greatly enhanced the effect of cognate ligand. Exogenous NGF induced greater phosphorylation of TrkA and AKT. This was associated with increased cell number in TrkA/p75 cells compared to TrkA cells (p<0.01), which was due to increased proliferation in TrkA/p75 cells (p<0.05), followed by differentiation. Exogenous BDNF also increased cell number in TrkB/p75 compared to TrkB cells (p<0.01), due to an increase in proliferation, but without differentiation. Coexpression of p75 also increased specificity of Trk-expressing cells to ligand. NT3-induced phosphorylation of TrkA and AKT was reduced in TrkA/p75 cells. NT3-induced phosphorylation of TrkB (as well as AKT and MAPK) was also reduced with p75 coexpression. Our results suggest that p75 plays an important role in enhancing both the sensitivity of Trk receptors to low levels of ligand, as well as increasing the specificity of Trks to their cognate ligands. It also enhances ligand-induced differentiation in TrkA/p75 but not TrkB/p75 cells.

Different effects of TrkA expression in neuroblastoma cell lines with or without MYCN amplification.

BACKGROUND Expression of the neurotrophin receptor TrkA is associated with a favorable prognosis in neuroblastoma (NB) and promotes growth inhibition and neuronal differentiation. Aggressive, MYCN-amplified NB tumors express little or no TrkA mRNA, suggesting that MYCN overexpression may inhibit TrkA expression. PROCEDURE To study the interactions of TrkA expression and MYCN amplification in NB, we stably expressed the TrkA receptor in the MYCN single copy cell lines SH-SY5Y and NB69 as well as in the MYCN amplified cell lines CHP134 and IMR5. RESULTS All four transfected cell lines demonstrated high TrkA expression and similar activation of the TrkA receptor and of mitogen-activated protein kinases as well as induction of immediate-early genes in response to nerve growth factor (NGF). Introduction of TrkA restored NGF responsiveness of SH-SY5Y and NB69 cells, as demonstrated by morphologic differentiation, growth inhibition, and enhanced survival in serum-free medium. However, no morphologic, growth, or survival responses to NGF were detected in MYCN-amplified CHP134 and IMR5 TrkA transfectants. CONCLUSIONS Thus, transfection of TrkA into MYCN amplified NB cell lines only partly restored the TrkA/NGF signaling pathway, suggesting additional inhibitory effects of MYCN overexpression on TrkA signaling.