Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis (UNSL), Ejército de los Andes 950, 5700 San Luis, Argentina; Instituto Multidisciplinario de Investigaciones Biológicas (IMIBIO-SL), CONICET, Argentina.
Instituto de Biología y Medicina Experimental de Cuyo (IMBECU), CONICET, Argentina.
Biochim Biophys Acta Mol Basis Dis. 2023 Oct;1869(7):166801. doi: 10.1016/j.bbadis.2023.166801. Epub 2023 Jul 6.
Over the last years, the incidence of melanoma, the deadliest form of skin cancer, has risen significantly. Nearly half of the melanoma patients exhibit the BRAF mutation. Although the use of BRAF and MEK inhibitors (BRAFi and MEKi) showed an impressive success rate in melanoma patients, durability of response remains an issue because tumor quickly becomes resistant. Here, we generated and characterized Lu1205 and A375 melanoma cells resistant to vemurafenib (BRAFi). Resistant cells (Lu1205R and A375R) exhibit higher IC (5-6 fold increase) and phospho-ERK levels and 2-3 times reduced apoptosis than their sensitive parents (Lu1205S and A375S). Moreover, resistant cells are 2-3 times bigger, display a more elongated morphology and have a modulation of migration capacity. Interestingly, pharmacological inhibition of sphingosine kinases, that prevents sphingosine-1-phosphate production, reduces migration of Lu1205R cells by 50 %. In addition, although Lu1205R cells showed increased basal levels of the autophagy markers LC3II and p62, they have decreased autophagosome degradation and autophagy flux. Remarkably, expression of Rab27A and Rab27B, which are involved in the release of extracellular vesicles are dramatically augmented in resistant cells (i.e. 5-7 fold increase). Indeed, conditioned media obtained from Lu1205R cells increased the resistance to vemurafenib of sensitive cells. Hence, these results support that resistance to vemurafenib modulates migration and the autophagic flux and may be transferred to nearby sensitive melanoma cells by factors that are released to the extracellular milieu by resistant cells.
在过去的几年中，黑色素瘤的发病率——最致命的皮肤癌形式——显著上升。近一半的黑色素瘤患者表现出 BRAF 突变。尽管 BRAF 和 MEK 抑制剂（BRAFi 和 MEKi）在黑色素瘤患者中的使用显示出令人印象深刻的成功率，但反应的持久性仍然是一个问题，因为肿瘤很快就会产生耐药性。在这里，我们生成并表征了对vemurafenib（BRAFi）产生耐药性的 Lu1205 和 A375 黑色素瘤细胞。耐药细胞（Lu1205R 和 A375R）表现出更高的 IC（增加 5-6 倍）和磷酸化 ERK 水平，以及比其敏感亲本（Lu1205S 和 A375S）低 2-3 倍的细胞凋亡。此外，耐药细胞体积大 2-3 倍，表现出更长的形态，并具有迁移能力的调节。有趣的是，抑制鞘氨醇激酶的药理学抑制作用可防止鞘氨醇-1-磷酸的产生，从而使 Lu1205R 细胞的迁移减少 50%。此外，尽管 Lu1205R 细胞显示出自噬标志物 LC3II 和 p62 的基础水平增加，但它们的自噬体降解和自噬流减少。值得注意的是，参与细胞外囊泡释放的 Rab27A 和 Rab27B 的表达在耐药细胞中显著增加（即增加 5-7 倍）。事实上，从 Lu1205R 细胞获得的条件培养基增加了敏感细胞对 vemurafenib 的耐药性。因此，这些结果表明对 vemurafenib 的耐药性调节迁移和自噬流，并可能通过耐药细胞释放到细胞外环境的因子转移到附近的敏感黑色素瘤细胞。
