PO.ET03.07 · 实验与分子治疗
通过抑制蛋白磷酸酶5（PP5）克服ccRCC中的药物耐药
Overcoming drug resistance in ccRCC through inhibition of protein phosphatase 5 (PP5)
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：Belzutifan是一种FDA批准的小分子抑制剂，用于治疗晚期透明细胞肾细胞癌（ccRCC）患者。Belzutifan抑制HIF2alpha，这是一种促进肿瘤血管生成和转移的转录因子。尽管ccRCC患者可能对belzutifan产生耐药，但目前尚无策略来对抗患者中的这种药物耐药。我们发现丝氨酸/苏氨酸蛋白磷酸酶5（PP5）的表达和活性在ccRCC中升高，促成其促存活作用。我们设计并开发了PP5的小分子抑制剂。本研究的目的是检验PP5抑制是否能在belzutifan耐药的ccRCC细胞中引起凋亡。
方法：通过用10μM belzutifan处理786-O细胞来建立belzutifan耐药的ccRCC细胞。一旦达到70%汇合，将细胞分传并再次用10μM belzutifan处理。重复此过程，直至细胞看起来健康并以与未经belzutifan处理的786-O相似的速率生长。在进行额外药物处理和/或蛋白提取前，将belzutifan从细胞中移除至少24小时。化合物P053是PP5的第二代小分子抑制剂。用1μM或10μM的P053处理belzutifan耐药的ccRCC细胞24小时，并通过免疫印迹评估凋亡标志物，检测切割的caspase-3和切割的PARP。
结果：第二代PP5抑制剂P053能够结合催化结构域并抑制PP5的磷酸酶活性。Belzutifan耐药的ccRCC细胞能够在10μM belzutifan存在下维持生长，且不显示任何凋亡激活的标志。然而，用1μM或10μM的P053处理耐药细胞诱导了凋亡，表现为切割的caspase-3和切割的PARP水平升高。
结论：用我们新型小分子抑制剂进行的PP5抑制保留了在belzutifan耐药细胞中引起凋亡的能力。这表明即使在产生belzutifan耐药后，PP5仍是晚期ccRCC中一个可行的治疗靶点。
查看英文原文 English abstract
BACKGROUND: Belzutifan is an FDA approved small molecule inhibitor used for the treatment of patients with advanced clear cell renal cell carcinoma (ccRCC). Belzutifan inhibits HIF2alpha, a transcription factor that promotes tumor angiogenesis and metastasis. Although ccRCC patients can potentially develop resistance to belzutifan there are currently no strategies to counteract this drug resistance in patients. We have identified that serine/threonine protein phosphatase-5 (PP5) expression and activity is elevated in ccRCC, contributing to its pro-survival role. We have designed and developed small molecule inhibitors of PP5. The objective of this study was to examine whether PP5 inhibition can cause apoptosis in belzutifan-resistant ccRCC cells.
METHODS: Belzutifan-resistant ccRCC cells were developed by treating 786-O cells with 10μM belzutifan. Once 70% confluent, the cells were split and treated with 10μM belzutifan again. This process was repeated until the cells appeared healthy and grew at a similar rate to 786-O without belzutifan treatment. Belzutifan was removed from the cells for at least 24 hours before additional drug treatments and/or protein extraction. Compound P053 is a second-generation small molecule inhibitor of PP5. Belzutifan-resistant ccRCC cells were treated with either 1μM or 10μM of P053 for 24 hours and apoptotic markers were evaluated by immunoblotting to examine cleaved caspase-3 and cleaved-PARP.
RESULTS: The second generation PP5 inhibitor P053 has the ability to bind to the catalytic domain and inhibit the phosphatase activity of PP5. Belzutifan-resistant ccRCC cells are able to maintain growth in the presence of 10μM belzutifan and do not display any hallmarks of activation of apoptosis. Treatment of belzutifan-resistant cells with either 1μM or 10μM of P053, however, induced apoptosis, as evidenced by elevated levels of cleaved caspase-3 and cleaved-PARP.
CONCLUSIONS: PP5 inhibition with our novel small molecule inhibitor retains the ability to cause apoptosis in belzutifan-resistant cells. This suggests PP5 is a viable therapeutic target in advanced ccRCC even after belzutifan resistance.
利益披露 Disclosure
S. J. Backe, None..
R. Sager, None.