Silencing E3 Ubiqutin ligase ITCH as a potential therapy to enhance chemotherapy efficacy in p53 mutant neuroblastoma cells

Abstract

P53 mutations are responsible for drug-resistance of tumour cells which impacts on the efficacy of treatment. Alternative tumour suppressor pathways need to be explored to treat p53- deficient tumours. The E3 ubiquitin ligase, ITCH, negatively regulates the tumour suppressor protein TP73, providing a therapeutic target to enhance the sensitivity of the tumour cells to the treatment. In the present study, two p53-mutant neuroblastoma cell lines were used as in vitro models. Using immunostaining, western blot and qPCR methods, we firstly identified that ITCH was expressed on p53-mutant neuroblastoma cell lines. Transfection of these cell lines with ITCH siRNA could effectively silence the ITCH expression, and result in the stabilization of TP73 protein, which mediated the apoptosis of the neuroblastoma cells upon irradiation treatment. Finally, in vivo delivery of the ITCH siRNA using nanoparticles to the neuroblastoma xenograft mouse model showed around 15-20% ITCH silencing 48 hours after transfection. Our data suggest that ITCH could be silenced both in vitro and in vivo using nanoparticles, and silencing of ITCH sensitizes the tumour cells to irradiation treatment. This strategy could be further explored to combine the chemotherapy/radiotherapy treatment to enhance the therapeutic effects on p53-deficient neuroblastoma.

Figure 1

Expression of ITCH and TP73 in neuroblastoma cell lines. (A) RT-PCR and the qPCR results of the expression in Kelly cells and BE2 cells, (B) immunostaining showing the expression of ITCH and TP73 at the protein level, scale bar = 25 µm.

Figure 2

Expression of integrin αv, β3 and β5 in neuroblastoma cells. (a) RT-PCR; (b) western blot and (c) immunostaining all showed the presence of these integrin molecules in the neuroblastoma cell lines, Kelly and BE2, scale bar = 25 µm.

Figure 3

Knockdown of ITCH in vitro using LipofectAMINE2000 (L2K) (A,B) or nanoparticles (C). (A) qPCR of ITCH mRNA (a and b) of Kelly cells transfected with different concentrations of ITCH siRNA (a) or using different amounts of L2K reagent (b). The cell viability in each transfection condition is indicated by %PI + cells after transfection (c,d). (B) Western blot showed the knockdown of ITCH protein by siRNA transfection in Kelly cells (B-a) or BE2 cells (B-b). L2K = L2K only. (C) Expression of ITCH mRNA (C-a and C-b) and ITCH protein (C-c) on Kelly cells after transfection of 100 nM ITCH siRNA with different nanoparticle formulations for 4 hours (C-a) or 48 hours (C-b). ITCH protein level is measured by western blot 48 hours after transfection (C-c).

Figure 4

Knockdown of ITCH causes the upregulation of TP73 (A) and induces apoptosis of Kelly cells upon irradiation. (A) Western blot showing silencing of ITCH protein and upregulation of TP73 after transfection of ITCH siRNA in Kelly cells. 1, 3, 5 and 7 are samples transfected with ITCH siRNA; 2, 4, 6 and 8 are samples transfected with irrelevant siRNA. D1, D2, D3 and D6 suggested days after transfection. (B) Apoptotic cells after irradiation of Kelly cells which have been transfected with ITCH siRNA (b) and irrelevant siRNA (a). Quantification of percentage of SubG1 cells within each treatment group (c).

Figure 5

ITCH expression in tumours 24 hours and 48 hours after ITCH siRNA treatment. (A) Relative ITCH expression in each treatment group. (B) Individual sample responses to the ITCH siRNA treatment at 24 hours or 48 hours. There were no differences between the treatment group and the control group 24 hours after treatment. There were, however, more samples with lower levels of ITCH expression in the treated group than the control group 48 hours after treatment.

Figure 6

Percentage of tumour samples of each treatment group which express ITCH mRNA relatively lower than 1, 0.9, 0.8 and 0.7, respectively.