Autophagy is associated with chemoresistance in neuroblastoma

Abstract

Background: Neuroblastoma (NB) is a frequent pediatric tumor characterized by a poor prognosis where a majority of tumors progress despite intensive multimodality treatments. Autophagy, a self-degradative process in cells, could be induced by chemotherapy and be associated with chemoresistance. The aim of this study was to determine whether: 1) autophagy is present in NB, 2) chemotherapy modified its levels, and 3) its inhibition decreased chemoresistance.

Methods: Immunohistochemical stainings were performed on samples from 184 NB patients in order to verify the expression of LC3B, a specific marker for autophagy, and Beclin 1, a positive regulator of autophagy. In addition, we performed an in vitro study with six NB cell lines and six drugs (vincristine, doxorubicin, cisplatin temozolomide, LY294002 and syrolimus). Inhibition of autophagy was performed using ATG5 knockdown cells or hydroxychloroquine (HCQ). Cell survival was measured using the MTT cell proliferation assay. Autophagy was detected by monodansylcadaverine, confocal microscopy and Western blot. In vivo study with tumor xenografts in NSG mice was performed.

Results: Our results have indicated that autophagy was present at low levels in NB and was not a prognostic factor, while Beclin 1 was highly expressed in children with poor NB prognosis. However, autophagy levels increased after chemotherapy in vitro and in vivo. Tumor progression was significantly decreased in mice treated with a combination of HCQ and vincristine.

Conclusions: Taken together, autophagy is present in NB, induced by chemotherapy and associated with chemoresistance, which is significantly reduced by its inhibition. Therefore, targeting autophagy represents a very attractive approach to develop new therapeutic strategies in NB.

Keywords: Autophagy; Chemoresistance; Hydroxychloroquine; Neuroblastoma.

Fig. 1

Evaluation of the level and the regulation of autophagy in NB and its correlation with apoptosis. A Autophagy was evaluated in TMA sections from NB tumor samples by immunohistochemistry using antibodies anti-LC3B (A.a,b) or anti-Beclin 1 (A.c). The expression levels of pmTOR (A.e) and pAKT (A.f), two autophagy-regulating proteins, were also studied on the same samples. Apoptosis was tested by TUNEL (A.d). Scale bars: 100 μm. B Immunoblotting analysis was performed on protein lysates from different frozen matched tumor samples with antibodies against either LC3B or Beclin 1 for autophagy and against cleaved caspase-3 for apoptosis. C The expression of LC3II and TUNEL positivity were semi-quantified according to the stage of tumors. D The ratio LC3II/LC3I and cleaved caspase-3 expression were evaluated by densitometry using tumor samples

Fig. 2

Chemotherapy induces autophagy in vitro and in vivo. The effect of chemotherapeutic agents on the autophagy of NB cell lines and tissues was analyzed. a NB-10 cells were treated with increasing concentrations of temozolomide, rapamycin, cisplatin, vincristine, doxorubicin or LY294002 for 24 h followed by MTT and MDC assays to measure cell viability and autophagy levels, respectively. Results are expressed as percentage of the corresponding control and represent mean ± SEM of 3 independent experiments (***: P < 0.001). b Cell viability and MDC specific autophagy were measured after IC50 treatment of different NB cell lines with chemotherapeutic agents. Results are expressed as percentage of the corresponding control and represent mean ± SEM of 3 independent experiments (***: P < 0.001). c Immunobloting analyses were performed on protein lysates from SK-N-SH and IGR-N91 cells treated with 0.1 to 10 μM of vincristine. Anti PARP-1, anti-LC3B, anti-p62, anti-mTOR, anti-pmTOR antibodies were used. β-actin was used as a loading control. d Tumor xenografts developed from SK-N-DZ or IGR-N91 cells were treated with cisplatin or vincristine. Slides were immunostained with anti-LC3B antibody. Scale bars: 100 μm

Fig. 3

Inhibition of autophagy sensitizes NB cells to chemotherapy. IGR-N91 cells were transduced with ATG5-shRNA lentivirus to knockdown ATG5 gene expression and thereby inhibit autophagy or with eGFP-shRNA, used as a control. a Protein immunoblotting using anti-ATG5 and anti-LC3B antibodies was performed to demonstrate ATG5 gene knockdown and LC3 expression on IGR-N91-transduced cells or not (WT). β-actin was used as a loading control. b A MDC test for autophagy was performed on ATG5 ^kd and control cells after treatment with increasing concentrations of vincristine. c Cell viability of ATG5 ^kd and control cells was measured with a MTT assay following treatment with increasing concentrations of vincristine, doxorubicin or cisplatin. Results are expressed as percentage of corresponding control and represent mean ± SEM of 4 independent experiments (*: P < 0.05, ***: P < 0.001)

Fig. 4

HCQ sensitizes NB cells to chemotherapy by inhibition of autophagy. a IGR-N91 and SK-N-SH cells were treated with 15 to 60 μM of HCQ and analyzed for LC3B, p62, mTOR, p-mTOR, AKT, pAKT, Beclin 1 protein expression by immunoblotting. β-actin was used as a loading control. b Cell viability of SK-N-DZ, IGR-N91 and IGR-NB8 was measured using an MTT assay pre and post HCQ treatment (30 μM). c Confocal microscopy analysis was done on transfected IGR-N91 cells with GFP-LC3 not treated or treated 7 h with HCQ 30 μM, vincristine 1 μM or the combination of the two drugs. d Autophagy in SK-N-DZ, IGR-N91 and IGR-NB8 cells was measured by an MDC assay pre and post HCQ treatment (30 μM). e SK-N-DZ cell viability was measured after vincristine or doxorubicin treatment combined or not with HCQ (30 μM). f The autophagic activity of SK-N-DZ cells was measured using a MDC agent after treatment with vincristine or doxorubicin, combined or not with HCQ (30 μM). Results are expressed as percentage of corresponding control and represent mean ± SEM of 4 independent experiments (*: P < 0.05, **: P < 0.01, ***: P < 0.001)

Fig. 5

Effect of autophagy inhibition by HCQ on tumors in vivo. Six NSG mice of each group received a s.c injection of 5x10⁶ IGR-N91 cells. When tumors reached 100 mm³, mice were administered vincristine (0.4 mg/kg/day), HCQ (60 mg/kg), an association of vincristine and HCQ or vehicle (saline) for 9 days. The tumor size of the four groups of mice was compared during this period