PO.ET03.08 · 实验与分子治疗
在Ras突变细胞中共靶向B-Raf和ACLY导致细胞活力协同丧失和凋亡
Cotargeting B-Raf and ACLY in Ras mutant cells leads to synergistic loss of cell viability and apoptosis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
Ras突变见于包括胰腺癌和结肠癌在内的多种肿瘤类型。位于Ras下游的是B-Raf激酶，其在黑色素瘤等肿瘤细胞类型中也常发生突变。B-Raf抑制剂维莫非尼（Vemurafenib）、达拉非尼（Dabrafenib）和恩考非尼（Encorafenib）在临床上与MEK抑制剂曲美替尼（Trametinib）、考比替尼（Cobimetinib）和比美替尼（Binimetinib）联合使用。这些药物共同抑制MAPK通路，但PI3K/AKT通路常上调，导致治疗耐药。AKT在包括代谢在内的众多细胞过程中发挥功能。事实上，AKT通过直接磷酸化并激活ATP-柠檬酸裂解酶（ACLY）来激活脂质生成。ACLY的表达和活性在多种癌症细胞类型中升高。一种新的ACLY抑制剂NDI-091143近来基于ACLY的结构被鉴定出来，并用于本研究。使用一组Ras突变细胞系来研究将ACLY抑制剂NDI-091143与BRAF抑制剂维莫非尼联合对细胞增殖和凋亡的影响。使用各药物单独将细胞活力降低25%的浓度，我们发现该组合在Ras突变的A375黑色素瘤细胞、MDA-MB-231乳腺癌细胞、MIA Paca-2胰腺癌细胞和HCT116结肠癌细胞中表现出细胞活力的协同降低。具有野生型Ras的癌细胞（如MeWo黑色素瘤和T47D乳腺癌）对该组合未显示协同的活力丧失。非转化细胞（如结肠上皮细胞CCD-18Co和正常胰腺细胞H6c7）不受维莫非尼与NDI-091143联合治疗所用相同浓度的影响。活力降低的机制似乎归因于凋亡的激活。所有四种Ras突变细胞系均显示切割型PARP表达增加以及内质网应激通路调节因子ATF4的激活。最后，利用对经维莫非尼加NDI-091143处理的HCT116细胞进行的Annexin V分析表明，该组合所刺激的凋亡依赖于Caspase-3活性。
查看英文原文 English abstract
Ras mutation is found in several tumor types including pancreatic and colon cancer. Downstream from Ras is the B-Raf kinase, also often found mutated in tumor cell types such as melanoma. B-Raf inhibitors Vemurafenib, Dabrafenib and Encorafenib are used clinically in combination with MEK inhibitors Trametinib, Cobimetinib and Binimetinib. Together, these agents serve to inhibit the MAPK pathway, but the PI3K/AKT pathway is often upregulated leading to treatment resistance. AKT functions in numerous cellular processes including metabolism. In fact, AKT activates lipogenesis by direct phosphorylation and activation of the enzyme ATP-Citrate Lyase (ACLY). ACLY expression and activity are elevated in various cancer cell types. A new inhibitor of ACLY, NDI-091143, was recently identified based on the structure of ACLY and was used in this study. A panel of Ras mutant cell lines was used to investigate the effect of combining the ACLY inhibitor NDI-091143 with the BRAF inhibitor Vemurafenib on cell proliferation and apoptosis. Using concentrations of each drug that reduced cell viability individually by 25%, we found that the combination exhibited synergistic reduction of cell viability in Ras mutant A375 melanoma cells, MDA-MB-231 breast cancer cells, MIA Paca-2 pancreatic cancer cells and HCT116 colon cancer cells. Cancer cells with WT Ras such as MeWo melanoma and T47D breast cancer showed no synergistic loss of viability in response to the combination. Non-transformed cells such as colon epithelial (CCD-18Co) and normal pancreatic cells (H6c7) are unaffected by the same concentrations used in the Vemurafenib and NDI-091143 combination treatment. The mechanism of reduction in viability appears to be due to activation of apoptosis. All four Ras mutant cell lines showed increased expression of cleaved PARP and the activation of the ER stress pathway regulator ATF4. Finally, the apoptosis stimulated by the combination was shown to be dependent on Caspase-3 activity using Annexin V analysis of HCT116 cells treated with Vemurafenib plus NDI-091143.
利益披露 Disclosure
N. A. Krucher, None..
B. D. Aierstok, None..
S. Bergesio, None..
G. J. Galgano, None..
M. E. Strecker, None.