Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors

Sanja Aveic, Marcella Pantile, Pierfrancesco Polo, Viktoryia Sidarovich, Marilena De Mariano, Alessandro Quattrone, Luca Longo, Gian Paolo Tonini

A growing field of evidence suggests the involvement of oncogenic receptor tyrosine kinases (RTKs) in cell transformation. Deregulated activity of RTKs in tumors can determine disease progression and therapeutic responses in several types of cancer, including neuroblastoma (NB). Therefore, RTKs targeting is a worthwhile challenge for the oncologists. Nevertheless, acquired resistance to RTK inhibitors (RTKi) remains a serious problem. Autophagy activation is among the possible obstacles for good efficacy of the therapy with RTKi. Under different treatment conditions we measured autophagic flux using immunoblot and immunofluorescence assays. Death induction was validated by trypan blue exclusion assay and FACS analysis (calcein-AM/propidium iodide). The NB cell lines SH-SY5Y and Kelly were used for the in vitro study. In order to define whether autophagy might be a limiting factor for the efficacy of RTKi in NB cells, we firstly checked its activation following the treatment with several RTKi. Next, we investigated the possibility to increase their therapeutic efficiency by combining RTKi with autophagy blocking agents in vitro. We exploited the effectiveness of three RTKi either alone or in combination with autophagy inhibitors (Chloroquine-CQ and Spautin-1). We demonstrated that autophagy induction was drug-dependent, and that its inhibition increased the anti-tumor activity of a single RTKi unevenly. We observed that the combined use of blocking agents which impair late autophagy events, such as CQ, and RTKi can be more effective with respect to the use of RTKi alone. In the present report, we assessed the conditions under which autophagy is activated during the use of different RTKi currently in the pre-clinical evaluation for NB. We summarized the achievements of combined RTK/autophagy inhibitors treatment as a promising approach to enhance the efficacy of RTKi in impairing tumor cells viability.