Medical Oncology Clinic, Free University of Brussels, 1000 Brussels, Belgium.
Int J Oncol. 2013 Sep;43(3):919-26. doi: 10.3892/ijo.2013.2008. Epub 2013 Jul 8.
Targeted therapy has shown high efficacy in the treatment of metastatic melanoma with impressive response rates. However, resistance appears after a few months, underlining the need for simultaneous multiple signalling pathway inhibition to provide a durable benefit. The aim of our study was to evaluate the possible synergistic effect of various protein kinase inhibitor combinations targeting SRC, MEK, PI3K or JAK on the survival of representative melanoma cell lines with WTNRAS/WTBRAF and harbouring the most frequent mutations (Q61LNRAS/WTBRAF or WTNRAS/V600EBRAF). By comparing IC50s and protein inhibition profiles, cell exposure to a single inhibitor for 3 days (condition 1) showed that both WTBRAF lines were at least 15-fold more sensitive to SRC inhibition while V600EBRAF cells were 30-fold more sensitive to MEK inhibition, confirming that the latter cells are largely dependent on the MAPK pathway for growth. Concomitant treatment for 3 days (condition 2) revealed an antagonistic effect between SRC and JAK inhibitors as compared to treatment by each inhibitor alone in all 3 lines, supporting that both SRC and JAK stimulate the STAT pathway. Finally, sequential cell exposure to inhibitors by pre-treatment with a single effector at non-toxic but effective on target inhibition concentrations for 7 days followed by the addition of each of the other inhibitors for 3 days (condition 3) showed that MEK, PI3K or JAK inhibitor acted in synergy with the SRC inhibitor in both wild-type and Q61LNRAS cells, suggesting that the first inhibitor could activate the SRC/STAT compensatory signalling pathway. In conclusion, a treatment strategy consisting in a sequential use of targeted inhibitors to first render melanoma cells more dependent on alternative compensatory pathways that should subsequently be inhibited, may enhance efficacy. By contrast, concomitant exposure to various combinations of inhibitors at different concentrations failed to produce such effect, further supporting the importance of both the duration of cell exposure to inhibitors and their sequential use.
靶向治疗在转移性黑色素瘤的治疗中显示出了很高的疗效，令人印象深刻的响应率。然而，几个月后就会出现耐药性，这突显了同时抑制多个信号通路以提供持久益处的必要性。我们的研究目的是评估针对 SRC、MEK、PI3K 或 JAK 的各种蛋白激酶抑制剂组合对具有 WTNRAS/WTBRAF 且携带最常见突变（Q61LNRAS/WTBRAF 或 WTNRAS/V600EBRAF）的代表性黑色素瘤细胞系存活的可能协同作用。通过比较 IC50 和蛋白抑制谱，细胞在单一抑制剂中暴露 3 天（条件 1）表明，WTBRAF 两条线对 SRC 抑制的敏感性至少高 15 倍，而 V600EBRAF 细胞对 MEK 抑制的敏感性高 30 倍，这证实了后者细胞对 MAPK 通路的生长依赖性很大。同时治疗 3 天（条件 2）显示，与每种抑制剂单独处理相比，在所有 3 条线中，SRC 和 JAK 抑制剂之间存在拮抗作用，支持 SRC 和 JAK 均刺激 STAT 通路。最后，通过在非毒性但有效靶抑制浓度下用单一效应物预处理细胞 7 天，然后再添加其他每种抑制剂 3 天（条件 3）进行顺序细胞暴露抑制剂，显示 MEK、PI3K 或 JAK 抑制剂与 SRC 抑制剂在 WT 和 Q61LNRAS 细胞中均具有协同作用，表明第一种抑制剂可以激活 SRC/STAT 补偿性信号通路。总之，一种治疗策略包括顺序使用靶向抑制剂，首先使黑色素瘤细胞对替代补偿途径更依赖，随后应抑制这些途径，这可能会增强疗效。相比之下，同时以不同浓度暴露于各种抑制剂组合未能产生这种效果，进一步支持了细胞暴露于抑制剂的持续时间及其顺序使用的重要性。
