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Comparative Study
. 2012 Oct;3(10):1169-81.
doi: 10.18632/oncotarget.600.

Targeting JNK-interacting-protein-1 (JIP1) sensitises osteosarcoma to doxorubicin

Jantine Posthumadeboer  1 , Pim W van Egmond, Marco N Helder, Renée X de Menezes, Anne-Marie Cleton-Jansen, Jeroen A M Beliën, Henk M W Verheul, Barend J van Royen, Gert-Jan J L Kaspers, Victor W van Beusechem
Affiliations
Comparative Study

Targeting JNK-interacting-protein-1 (JIP1) sensitises osteosarcoma to doxorubicin

Jantine Posthumadeboer et al. Oncotarget. 2012 Oct.

Abstract

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Despite aggressive therapy, survival outcomes remain unsatisfactory, especially for patients with metastatic disease or patients with a poor chemotherapy response. Chemoresistance contributes to treatment failure. To increase the efficacy of conventional chemotherapy, essential survival pathways should be targeted concomitantly. Here, we performed a loss-of-function siRNA screen of the human kinome in SaOS-2 cells to identify critical survival kinases after doxorubicin treatment. Gene silencing of JNK-interacting-protein-1 (JIP1) elicited the most potent sensitisation to doxorubicin. This candidate was further explored as potential target for chemosensitisation in OS. A panel of OS cell lines and human primary osteoblasts was examined for sensitisation to doxorubicin using small molecule JIP1-inhibitor BI-78D3. JIP1 expression and JIP1-inhibitor effects on JNK-signalling were investigated by Western blot analysis. JIP1 expression in human OS tumours was assessed by immunohistochemistry on tissue micro arrays. BI-78D3 blocked JNK-signalling and sensitised three out of four tested OS cell lines, but not healthy osteoblasts, to treatment with doxorubicin. Combination treatment increased the induction of apoptosis. JIP1 was found to be expressed in two-thirds of human primary OS tissue samples. Patients with JIP1 positive tumours showed a trend to inferior overall survival. Collectively, JIP1 appears a clinically relevant novel target in OS to enhance the efficacy of doxorubicin treatment by means of RNA interference or pharmacological inhibition.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1. siRNA library screen of the human kinome identifies enhancers of doxorubicin response in OS
(A) Schematic overview of the set-up of the screens performed with pools of 4 siRNAs against 788 human kinases and kinase-associated genes in SaOS-2 cells. (B) Screen results of the 10 selected candidate hits showing the effects of gene-silencing only (grey bars) versus gene-silencing + doxorubicin treatment (black bars) on cell viability. Bars represent the average cell viability measured in the 3 screens; error bars indicate standard deviations (SD). (C) Pie chart summarizing the secondary screen results. The chart shows the number of separate siRNA duplexes of 4 individual siRNAs tested per gene, that reproduced the doxorubicin-sensitising phenotype of the pooled siRNAs per gene. (Bar graphs for the separate siRNA duplexes are provided in panel D for JIP1 and Supplementary Figure S1 for the other genes). (D) Confirmation of the doxorubicin-sensitising phenotype with siRNAs against JIP1. Cells were transfected with the indicated siRNA duplex and cultured in the presence (black bars) or absence (grey bars) of doxorubicin at IC20 concentration. Bars represent results from an experiment performed in triplicate; error bars indicate SD..
Figure 2
Figure 2. siRNA targeting JIP1 reduces JIP1 mRNA and protein expression in SaOS2 cells and causes increased sensitivity to doxorubicin
(A) JIP1 mRNA silencing after transfection of JIP1 siRNA duplexes, analysed by RT-qPCR. JIP1 mRNA expression levels were normalised to GAPDH mRNA levels for each sample. Bars represent experiments performed in triplicate, error bars indicate SD. Results could not be obtained with JIP1 siRNA duplex #1 due to major cytotoxicity upon transfection of this siRNA. (B) Western blot analysis of JIP1 protein depletion at the indicated time-points after transfection of JIP1 siRNA duplex #2. (C) Dose-response curve of cells treated with doxorubicin (closed squares) and cells treated with doxorubicin after JIP1 gene silencing using JIP1 siRNA duplex #2 (open triangles). Results were obtained in an experiment performed in triplicate; error bars indicate SD. The JIP1 silenced cells show a significantly increased sensitivity to doxorubicin treatment (student's t-test at IC50; p < 0.0001).
Figure 3
Figure 3. JIP1 inhibition sensitises OS cells but not normal osteoblasts to doxorubicin-induced apoptosis
(A) Cells were treated in triplicate with doxorubicin (closed squares) or with doxorubicin and JIP1-inhibitor BI-78D3 (open triangles) and cell viability was determined four days later. Sigmoidal dose-response curves were created and IC50 values were calculated. The sensitising effect of JIP1 inhibition to doxorubicin treatment is significant in three out of four OS cell lines, (student's t-test at IC50; U2OS, p = 0.19, n.s; SaOS-2, p < 0.05; LM7 and MG-63, p < 0.0001). Human primary osteoblasts Hum63 and Hum65 do not show sensitisation to doxorubicin treatment in the presence of the JIP1-inhibitor (student's t-test at IC50; p = 0.459 and p = 0.417 respectively). (B) Caspase activation in OS cells treated with doxorubicin (grey bars) or with doxorubicin and JIP1-inhibitor BI-78D3 (black bars). White bars represent the control condition, which was set to 1. Bars represent an experiment performed in triplicate, error bars indicate SD. After combination treatment, caspase activity is distinctly higher in SaOS2, LM7 and MG-63 compared to doxorubicin treatment alone (student's t-test; LM7: **, p < 0.01; MG-63: *, p < 0.05; SaOS2, p = 0.08). In U2OS cells, caspase activity is comparable with and without JIP1 inhibition (p = 0.48).
Figure 4
Figure 4. JIP1-inhibitor BI-78D3 reduces JIP1/p-JNK complexes in doxorubicin treated OS cells
(A) Western blot analysis of baseline JIP1 levels in human OS cell lines and human primary osteoblast culture Hum71. All OS cell lines exhibit higher JIP1 levels than the human primary osteoblasts. (B) Immunoprecipitation and Western blot analysis of the JIP1/JNK activation module. LM7 and U2OS cells were treated with doxorubicin either in the presence or absence of BI-78D3. Whole cell lysates were analysed for JIP1 expression with β-actin serving as control for equal loading. In addition, cell lysates were immunoprecipitated with an anti-JIP1 antibody and Western blot analysis was done with an anti-p-JNK antibody. Doxorubicin-treated LM7 cells show an increased expression level of p-JNK compared to untreated cells, whereas in U2OS there is a slight decrease in p-JNK after doxorubicin treatment. After concurrent treatment with BI-78D3, p-JNK expression levels are diminished, indicative of inhibition of JIP1 scaffold function.
Figure 5
Figure 5. Kaplan-Meier overall survival analysis of OS patients with JIP1 positive or negative tumours
Kaplan-Meier survival plot showing the cumulative survival of patients suffering from localised OS. Patients were divided into groups of patients with JIP1 positive tumours (55 samples) and those with JIP1 negative tumours (16 samples). The observed difference in cumulative survival between the two groups shows a trend towards inferior survival outcomes for patients with positive JIP1 staining (p = 0.056).
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