Resistance to sunitinib in renal clear cell carcinoma results from sequestration in lysosomes and inhibition of the autophagic flux

Metastatic renal cell carcinomas (mRCC) are highly vascularized tumors that are a paradigm for the treatment with antiangiogenesis drugs targeting the vascular endothelial growth factor (VEGF) pathway. The available drugs increase the time to progression but are not curative and the patients eventually relapse. In this study we have focused our attention on the molecular mechanisms leading to resistance to sunitinib, the first line treatment of mRCC. Because of the anarchic vascularization of tumors the core of mRCC tumors receives only suboptimal concentrations of the drug. To mimic this in vivo situation, which is encountered in a neoadjuvant setting, we exposed sunitinib-sensitive mRCC cells to concentrations of sunitinib below the concentration of the drug that gives 50% inhibition of cell proliferation (IC50). At these concentrations, sunitinib accumulated in lysosomes, which downregulated the activity of the lysosomal protease CTSB (cathepsin B) and led to incomplete autophagic flux. Amino acid deprivation initiates autophagy enhanced sunitinib resistance through the amplification of autolysosome formation. Sunitinib stimulated the expression of ABCB1 (ATP-binding cassette, sub-family B [MDR/TAP], member 1), which participates in the accumulation of the drug in autolysosomes and favor its cellular efflux. Inhibition of this transporter by elacridar or the permeabilization of lysosome membranes with Leu-Leu-O-methyl (LLOM) resensitized mRCC cells that were resistant to concentrations of sunitinib superior to the IC50. Proteasome inhibitors also induced the death of resistant cells suggesting that the ubiquitin-proteasome system compensates inhibition of autophagy to maintain a cellular homeostasis. Based on our results we propose a new therapeutic approach combining sunitinib with molecules that prevent lysosomal accumulation or inhibit the proteasome.

转移性肾细胞癌（metastatic renal cell carcinoma，mRCC）是一类高血管化实体瘤，是靶向血管内皮生长因子（vascular endothelial growth factor，VEGF）通路的抗血管生成药物治疗的经典范式。目前临床可用的此类药物可延长患者的无进展生存期，但无法实现根治，患者最终仍会出现肿瘤复发。本研究聚焦于舒尼替尼（sunitinib，mRCC的一线治疗药物）耐药相关的分子机制。由于肿瘤血管生成紊乱，mRCC实体瘤的核心区域仅能接收到次优浓度的药物。为模拟新辅助治疗场景中体内的这一药物暴露情况，我们将对舒尼替尼敏感的mRCC细胞暴露于低于半数细胞增殖抑制浓度（half maximal inhibitory concentration，IC50）的舒尼替尼浓度环境中。在该浓度条件下，舒尼替尼会在溶酶体中蓄积，下调溶酶体蛋白酶CTSB（cathepsin B，组织蛋白酶B）的活性，并引发不完全自噬流。氨基酸剥夺可诱导自噬发生，并通过扩增自噬溶酶体的形成进一步增强肿瘤细胞对舒尼替尼的耐药性。舒尼替尼可上调ABCB1（ATP结合盒转运蛋白B亚家族成员1，ATP-binding cassette, sub-family B [MDR/TAP], member 1）的表达，该蛋白参与药物在自噬溶酶体中的蓄积，并促进药物的细胞外排。使用埃克拉达（elacridar）抑制该转运蛋白，或采用亮氨酰-亮氨酰-O-甲基酯（Leu-Leu-O-methyl，LLOM）实现溶酶体膜通透化，均可使对高于IC50浓度舒尼替尼产生耐药的mRCC细胞恢复药物敏感性。蛋白酶体抑制剂同样可诱导耐药细胞死亡，这表明泛素-蛋白酶体系统可代偿自噬抑制作用以维持细胞稳态。基于本研究结果，我们提出一种全新的治疗策略：将舒尼替尼与可阻断溶酶体药物蓄积或抑制蛋白酶体的分子联合使用。