Oncotarget. 2016 Feb 2; 7(5): 5646–5663.
Combating autophagy is a strategy to increase cytotoxic effects of novel ALK inhibitor entrectinib in neuroblastoma cells
Sanja Aveic,1 Marcella Pantile,1 Anke Seydel,1 Maria Rosaria Esposito,1 Carlo Zanon,1 Gary Li,2 and Gian Paolo Tonini1
1 Neuroblastoma Laboratory, Pediatric Research Institute, Città della Speranza, Padua, Italy
2 Ignyta Inc., San Diego, California, USA
Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells.
Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03–5 μM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67, and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib and demonstrated strong activation of autophagy in SH-SY5YF1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate and PARP protein cleavage in SH-SY5YF1174L cells.
In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib.
Receptor tyrosine kinases (RTKs) are transmembrane glycoproteins that transmit the signals from the extracellular space inside the cell. Thanks to their intrinsic enzymatic features, RTKs basically trigger a series of phosphorylation after the interaction with their extracellular signaling molecules. This, in turn, leads to the propagation of the initial message that concludes by the regulation of some of the numerous cell processes such as growth, migration, differentiation, survival, apoptosis, etc. The anaplastic lymphoma kinase (ALK) is RTK found deregulated in approximately 8-10% of all Neuroblastomas, one of the most common cancers that develops during childhood (1). It has been shown that deregulated ALK assures proliferative advantages to Neuroblastoma cells, giving a negative impact over prognosis of Neuroblastoma patients (2). Due to a significant role that ALK plays in Neuroblastoma tumorigenesis, but also other forms of malignancies, such as non-small cell lung cancer, numerous ALK antagonists have been developed and many of them proposed for the treatment of the patient bearing ALK-mutation. However, the most recent anti-ALK therapy showed certain restrictions in efficiency since malignant cells were able to turn on defence mechanisms, acquiring resistance against these inhibitors. One of the mechanisms that can provoke the resistance and impede the efficacy of anti-cancer drugs is autophagy. This protective process has been found along with low efficiency of RTK-inhibitors in tumour treatments as well (3). Having in mind a frequent connection between resistance to RTK-inhibitors and autophagy activation, we hypothesised that in case of Neuroblastoma cells, autophagy induction could stand for the impaired efficiency of Entrectinib, new ALK inhibitor proposed by Ignyta.
Beside examining the impact of Entrectinib over cell survival and death, showing a potent activity of this drug against ALK-amplified NB1 cell line, we were particularly interested in understanding the mechanism that underlies a low effectiveness of Entrectinib in ALKF1174L bearing Neuroblastoma cells. Therefore, we inquired the role of autophagy in observed conflict with Entrectinib treatment of ALK-mutated Neuroblastoma cells’, putting the light on the ALKF1174L cells particularly. Data that we obtained in our in vitro studies permitted us to distinguish a correlation between a level of drug’s inefficiency and autophagy activation, which was cell type specific. Importantly, autophagy impediment by Chloroquine (CQ), an antimalarial drug, led to important improvement of Entrectinib efficiency in terms of increased mortality of Neuroblastoma cells. This result is important since it promises a possibility to use combined treatment with anti-ALK and anti-autophagy compounds (e.g. CQ) in a synergy, improving therapy outcome. This certainly does not have to be the case to anti-ALK only but more likely to other RTK inhibitors as well, for which similar troublesome have been faced during their preclinical and clinical testing.
Figure 1. Autophagy activation by Entrectinib in ALKF1174L NB cell line. Transient transfection with GFP-LC3 plasmid was performed by Effectene Transfection Reagent (Qiagen) according to the manufacturer procedure. In total, 2 μg of plasmid was used and 24h post-transfection 5 μM of Entrectinib or DMSO was added for additional 24h. DAPI was used to mark cells’ nuclei. The localization of GFP-LC3 within SH-SY5YF1174L transfected cells was detected by fluorescence microscope. The Entrectinib treatment provoked vesicle formation in GFP-LC3-transfected cells, but not in DMSO treated controls. This images implied that LC3 was engaged in autophagosome formation during treatment with Entrectinib sustaining autophagy activation.
The importance of the study:
Even though the treatments with RTK inhibitors have been connected with autophagy activation for several types of malignancies this was not observed for Neuroblastoma till now. By investigating a newly proposed anti-ALK compound, Entrectinib, we delineated for the first time in Neuroblastoma that autophagy was a possible cause of weaken efficacy of this compounds in in vitro systems in which we would expect it to be highly active and successful. More precisely, we delineated the autophagy as a protective mechanism that was activated in ALK-mutated Neuroblastoma cells during administration of Entrectinib giving them a survival advantages during treatment. Numerous reports indicate the activation of autophagy as a protective mechanism that we have to keep in mind during treatment of cancers with deregulated RTKs activities. Therefore, several clinical trials in which the investigation of the RTK inhibitors in combination with chloroquine (CQ), or hydroxychloroquine (HCQ) in cancer patients are ongoing (4). According to the preclinical studies, it is reasonable to expect that the chemical inhibition of autophagy in combination with currently used therapy regiments adopted for Neuroblastoma patients might improve the results obtained when ALK-inhibitors are used alone. Moreover, if translated to other malignancies with deregulated RTKs, we might say that the co-inhibition of autophagy and deregulated RTK might be of crucial importance for an improved effectiveness of current treatment against diverse malignancies. We would like to focus current results on a possible link between RTK inhibitors and autophagy activation as a possible cause of low therapy effectiveness observed during administration of these drugs and combined treatments as a potential strategy for increased success.
Figure 2. Cell death validation after co-treatment with Entrectinib and Chloroquine. To determine the percentage of cell death provoked by a single treatment with Entrectinib (1 μM) or in combination with CQ (50 μM), we used flow cytometery (Cytomics FC500) and the Apodirect Kit (BD Bioscience). By TUNEL assay we distinguished a portion of dying SH-SY5Y cells after treatment with Entrectinib alone or in combination with CQ. Synergic activity between Entrectinib and CQ is demonstrated on the graph as an increase of the pick signal toward right side of the panel.
- Berry T, Luther W, Bhatnagar N, Jamin Y, Poon E, Sanda T, Pei D, Sharma B, Vetharoy WR, Hallsworth A, Ahmad Z, Barker K, Moreau L, et al. The ALK(F1174L) Mutation Potentiates the Oncogenic Activity of MYCN in Neuroblastoma. Cancer Cell 2012; 22:117–130.
- Passoni L, Longo L, Collini P, Coluccia AM, Bozzi F, Podda M, Gregorio A, Gambini C, Garaventa A, Pistoia V, Grosso F, Tonini GP, Cheng M, et al. Mutation in dependent anaplastic lymphoma kinase overexpression in poor prognosis neuroblastoma patients. Cancer Res 2009;69:7338–7346.
- Li Y, Lam S, Mak JC, Zheng C, Ho JC. Erlotinib-induced autophagy in epidermal growth factor receptor mutated non-small cell lung cancer. Lung Cancer. 2013;81:354–61. Cufí S, Vazquez-Martin A, Oliveras-Ferraros C, Corominas-Faja B, Cuyàs E, López-Bonet E, et al. The anti-malarial chloroquine overcomes primary resistance and restores sensitivity to trastuzumab in HER2-positive breast cancer. Sci. Rep. 2013;3:2469.
- Yang ZJ, Chee CE, Huang S, Sinicrope F. The role of autophagy in cancer: therapeutic implications. Mol. Cancer Ther. 2011;10:1533–1541.
Realization of this project was supported by Fondazione Italiana alla Lotta al Neuroblastoma.
Sanja Aveic, PhD
Laboratorio di Neuroblastoma
Fondazione Istituto di Ricerca Pediatrica Città della Speranza
Corso Stati Uniti, 4
Tel. +39 049 8215488