INHIBITION OF AUTOPHAGY INCREASES THE RESPONSE TO SUNITINIB IN CCRCC CELL LINES

Introduction: Renal cell carcinoma (RCC) represents about 3% of all cancers and is the kidney malignancy with the highest mortality rate of urinary neoplasms. The most common subtype of RCC is clear cell RCC (ccRCC) that accounts for 70-80% of RCC cases (1, 2). One third of cases presents metastasis at diagnosis with a 30% of disease recurrence for RCC patients undergoing surgery. Moreover, treatment with tyrosine kinase inhibitors in RCC subjects with advanced disease has not shown any advantage on overall survival (1). Therefore, research on new therapeutic targets focuses on molecules that can enhance therapy response. This is a crucial point to improve the management of RCC patient. The kidney cancer progression could be induced by the activation of autophagy, the biological process used by cancer cells to produce energy in hypoxic and acidic environment. Thus, the use of autophagy inhibitors could be considered as a novel antitumor therapy (3). In addition, there is emerging evidence that the use of autophagy inhibitors could sensitize cancer cells to anticancer drugs. Therefore, the combination of both tyrosine kinase and autophagy inhibitors could improve therapy response in ccRCC patients. Materials and Methods: Combined treatment with the autophagy inhibitor (Chloroquine) and anti-tyrosine kinase (Sunitinib) was performed in two different ccRCC cell lines (KJ29 and Caki-2). Cell growth was analyzed by Cell Tyter System (Promega, Madison, WI, USA), culturing cells in presence of both Chloroquine and Sunitinib for 24 and 48 h. Cell density after different treatments of ccRCC cells was evaluated by using a phase-contrast microscope at 10× magnification. Apoptosis was analyzed by the Hoechst method. Apoptotic nuclei were observed by a fluorescence microscope at 50× magnification after cell treatment with chloroquine and sunitinib. Results and Discussion: We observed that the upregulation of miR501-5p in ccRCC tissues is associated with a poor prognosis for ccRCC patients (2). Moreover, the overexpression of this miR induced the activation of autophagy in ccRCC cells. Cancer cells might use autophagy to trap and destroy molecular drugs by lysosomal vesicles. Therefore, the inhibition of autophagy should restore drug response. Consistently, we have observed that pre-treatment with Chloroquine, an autophagy inhibitor, in KJ29 ccRCC cells potentiated the Sunitinib-induced inhibition of cell growth compared to Sunitinib used alone. This finding was confirmed treating Caki-2, another ccRCC cell line, with both Chloroquine and Sunitinib. Moreover, the double treatment with these molecules reduced cell density compared to Chloroquine or Sunitinib, individually applied. The reduction of cell proliferation induced by the inhibition of both autophagy and tyrosine kinase receptors is associated with stimulation of apoptosis. In fact, the formation of apoptotic nuclei in double treated ccRCC cells, compared to those treated with the single compound, was observed. These results show that the inhibition of autophagy increases the efficacy of Sunitinib by the activation of apoptosis in ccRCC cells. Conclusion: These data suggest that the double treatment with both autophagy and tyrosine kinase inhibitors could be considered as a new therapeutic approach for the treatment of ccRCC patients. 1 Greef B and Eisen T: Medical treatment of renal cancer: new horizons. Br J Cancer 115: 505-516, 2016. 2 Mangolini A, Bonon A, Volinia S, Lanza G, Gambari R, Pinton P, Russo GR, Del Senno L, Dell'Atti L and Aguiari G: Differential expression of microRNA501-5p affects the aggressiveness of clear cell renal carcinoma. FEBS Open Bio 4: 952-965, 2014. 3 Kimura T, Takabatake Y, Takahashi A and Isaka Y: Chloroquine in cancer therapy: a doubleedged sword of autophagy. Cancer Res 73: 3-7, 2013.