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. 2010 Mar;120(3):850-8.
doi: 10.1172/JCI41013. Epub 2010 Feb 15.

Neurotrophin-3 production promotes human neuroblastoma cell survival by inhibiting TrkC-induced apoptosis

Jimena Bouzas-Rodriguez  1 , Jorge Ruben Cabrera, Céline Delloye-Bourgeois, Gabriel Ichim, Jean-Guy Delcros, Marie-Anne Raquin, Raphaël Rousseau, Valérie Combaret, Jean Bénard, Servane Tauszig-Delamasure, Patrick Mehlen
Affiliations

Neurotrophin-3 production promotes human neuroblastoma cell survival by inhibiting TrkC-induced apoptosis

Jimena Bouzas-Rodriguez et al. J Clin Invest. 2010 Mar.

Abstract

Tropomyosin-related kinase receptor C (TrkC) is a neurotrophin receptor with tyrosine kinase activity that was expected to be oncogenic. However, it has several characteristics of a tumor suppressor: its expression in tumors has often been associated with good prognosis; and it was recently demonstrated to be a dependence receptor, transducing different positive signals in the presence of ligand but inducing apoptosis in the absence of ligand. Here we show that the TrkC ligand neurotrophin-3 (NT-3) is upregulated in a large fraction of aggressive human neuroblastomas (NBs) and that it blocks TrkC-induced apoptosis of human NB cell lines, consistent with the idea that TrkC is a dependence receptor. Functionally, both siRNA knockdown of NT-3 expression and incubation with a TrkC-specific blocking antibody triggered apoptosis in human NB cell lines. Importantly, disruption of the NT-3 autocrine loop in malignant human neuroblasts triggered in vitro NB cell death and inhibited tumor growth and metastasis in both a chick and a mouse xenograft model. Thus, we believe that our data suggest that NT-3/TrkC disruption is a putative alternative targeted therapeutic strategy for the treatment of NB.

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Figures

Figure 1
Figure 1. NT-3 is expressed in a large fraction of stage 4 NBs.
(A) NT-3 expression and NT-3/TrkC ratio measured by Q-RT-PCR on total RNA from tumors from a total of 86 stage 4 NB patients. The percentage of tumors expressing NT-3 at more than 2 fold of the value corresponding to the median is indicated. NT-3-low, tumors with low levels of NT-3 expression; NT-3-high, tumors with high levels of NT-3 expression. (B) Representative NT-3 immunohistochemistry on a tumor biopsy and bone marrow dissociated cells from low (left panel) and high (right panel) NT-3–expressing stage 4 patients, corresponding to the dotted gray arrow and black arrow in A, respectively. (C) Representative NT-3 immunohistochemistry on CLB-Ge2, SHEP-CLB, CLB-VolMo, and IMR32 cells. CLB-Ge2 and SHEP-CLB NT-3 immunostaining, when an excess of recombinant NT-3 (r-NT-3) is added with primary antibody, is also shown. Note that the 4 top panels show immunohistochemistry performed in absence of membrane permeabilization (w/o), while the immunohistochemistry shown in 2 bottom panels was performed after cell permeabilization with Triton X-100 (w). (B and C) Insets depict control without primary antibody. Original magnification, ×32.
Figure 2
Figure 2. Disruption of NT-3 autocrine loop triggers NB cell death.
(A) NT-3 immunostaining on the CLB-Ge2 cell line 24 hours after transfection with scrambled siRNA (siRNA scr) or with NT-3 siRNA (siRNA NT-3). Insets depict control without primary antibody. Original magnification, ×32. (B and C) Cell death induction in CLB-Ge2, CLB-VolMo, SHEP-CLB, and IMR32 cell lines was quantified after transfection with either scrambled siRNA or a mix of 3 siRNAs targeting NT-3, using relative caspase-3 activity assay (B) or Toxilight assay (C). (D and E) Cell death induction in CLB-Ge2, CLB-VolMo, or IMR32 cell lines was quantified in cells treated with anti-TrkC blocking antibody (α TrkC) or without (control) anti-TrkC antibody, using relative caspase-3 activity assay (D) or TUNEL assay (E). For the TUNEL assay, a representative labeling of TUNEL-positive cells is shown (top panel, control cells; bottom panel, cells treated with anti-TrkC blocking antibody). Original magnification, ×20. (F) Effect of anti-TrkC blocking antibody on stage 4 NB. Tumoral cells were directly dissociated from the surgical biopsy and were plated for 24 hours in presence (+) or in absence (-) of treatment. (B–F) Data represent mean ± SEM. *P < 0.05, 2-sided Mann-Whitney test, compared with control.
Figure 3
Figure 3. NT-3/TrkC interference promotes TrkC proapoptotic activity.
(A) CLB-Ge2 cells were transfected with either empty vector or with a plasmid encoding the dominant-negative TrkC-IC D641N and treated for 24 hours with or without anti-TrkC blocking antibody Cell death was monitored by TUNEL labeling of cells plated on slides. The control panel shows TrkC-IC D641N by Western blots using anti-TrkC antibody (bottom panel). Representative images are shown. Original magnification, ×20. (B) The efficacy of TrkC siRNA was evaluated by Western blot on nonexpressing TrkC 13.S.24 olfactive neuroblasts. Cells were transfected either with empty vector or with uncleavable TrkC D945N D641N double mutant that does not trigger apoptosis, and with scrambled siRNA or TrkC siRNA (siRNA TrkC). (C) Cell death induction in the CLB-Ge2 cell line was quantified after transfection with either scrambled siRNA (siScr), TrkC siRNA (siTrkC), NT-3 siRNA (siNT-3), or a mix of TrkC and NT-3 siRNA, using relative caspase-3 activity assay. (D) Phospho-Akt and phospho-Erk levels of CLB-Ge2 cells were monitored by Western blot after 16 hours of treatment with 2 μg/ml anti-TrkC blocking antibody 20 nM Ly29402, 100 nM U0126, or 100 ng/ml NT-3, in absence of serum. (E) Detection of TrkC cleavage band (20 kDa, indicated by the arrow) by Western blot, using an anti-TrkC antibody on cells treated (or not) with anti-TrkC blocking antibody, with or without the general caspase inhibitor BAF. (A and C) Data represent mean ± SEM. *P < 0.05, 2-sided Mann-Whitney test, compared with control.
Figure 4
Figure 4. Blocking NT-3/TrkC inhibits NB growth and dissemination.
(A) Schematic representation of the experimental chick model. IMR32 or CLB-Ge2 cells were grafted in CAM at day 10, and anti-TrkC blocking antibody or an isotypic antibody (control antibody) was added on day 11 and day 14. Tumors and lungs were harvested on day 17. (B–D) Effect of anti-TrkC blocking antibody on primary tumor growth and apoptosis. (B) Representative images of CLB-Ge2 primary tumors formed on nontreated CAM or CAM treated either with an isotypic antibody (control antibody) or with anti-TrkC blocking antibody. Scale bar: 2 μm. (C) Representative images of TUNEL-positive cells in the respective primary tumors described in B. Scale bar: 100 μm. (D) Quantitative analysis showing the primary tumor size relative to nontreated tumors. (E) Effect of anti-TrkC blocking antibody on lung metastasis. The percentage of embryos with lungs invaded by IMR32 or CLB-Ge2 cells after 2 intratumoral injections (day 11 and day 14) of either anti-TrkC blocking antibody, an isotypic antibody, or nontreated. (F) Same as for D except that NT-3, TrkC, scrambled, or NT-3 and TrkC siRNA were injected on a chorioallantoic vessel. Relative primary tumor size is presented. (D and F) Data represent mean ± SEM. *P < 0.05, **P < 0.01, 2-sided Mann-Whitney test, compared with nontreated tumors. (E) **P < 0.01, χ2 test.
Figure 5
Figure 5. Disruption of NT-3 autocrine loop inhibits NB tumor growth.
(A) The volume of SHEP-CLB engrafted tumors was measured during treatment with either NT-3 siRNA or scrambled siRNA. Mice were injected intraperitoneally with 3 μg siRNA 3 times per week. Mean tumor volume is indicated. (B) SHEP-CLB tumors were dissected after 18 days of treatment. Final tumor weight and representative pictures are shown. Scale bar: 2 mm. Data represent mean ± SEM. **P < 0.01, ***P < 0.001, 2-sided Mann-Whitney test, compared with scrambled siRNA–treated mice.
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References

    1. Levi-Montalcini R, Angeletti PU. Essential role of the nerve growth factor in the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro. Dev Biol. 1963;7:653–659. doi: 10.1016/0012-1606(63)90149-0. - DOI - PubMed
    1. Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci. 2001;24:677–736. doi: 10.1146/annurev.neuro.24.1.677. - DOI - PMC - PubMed
    1. Nakagawara A. Trk receptor tyrosine kinases: a bridge between cancer and neural development. Cancer Lett. 2001;169(2):107–114. doi: 10.1016/S0304-3835(01)00530-4. - DOI - PubMed
    1. Porter AC, Vaillancourt RR. Tyrosine kinase receptor-activated signal transduction pathways which lead to oncogenesis. Oncogene. 1998;17(11 Reviews):1343–1352. - PubMed
    1. Xu X, Tahan SR, Pasha TL, Zhang PJ. Expression of neurotrophin receptor Trk-C in nevi and melanomas. J Cutan Pathol. 2003;30(5):318–322. doi: 10.1034/j.1600-0560.2003.00068.x. - DOI - PubMed

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