Background

Deficiency of folliculin (FLCN) may lead to renal cell carcinoma (RCC) in patients with Birt–Hogg–Dubé (BHD) disease. In this study, we investigated the cytotoxicity induced by PARP inhibitor olaparib in FLCN deficient RCC cells, and the interaction between FLCN and BRCA1 A complex-regulated DNA repair pathway.

Methods and Materials

FLCN expressing (ACHN and UOK257-F) and FLCN deficient (ACHN-2 and UOK257) cell lines were used in this research. Cell viability was detected by clonogenic assay and MTT assay. Flow cytometry and TUNEL assay were used to detect apoptosis. Autophagy in cells was measured by MDC assay, western blot, and transmission electron microscopy. Co-immunoprecipitation, immunofluorescence and western blot experiments were performed to determine the interaction between FLCN protein and BRCA1 A complex. The in vivo experiments were performed in a xenograft model by inoculating UOK 257 in nude mice.

Results

RCC cells with FLCN protein deficiency were more sensitive to olaparib treatment than the cells with FLCN expression. Olaparib treatment led to more severe autophagy and apoptosis in FLCN deficient ACHN-2 and UOK257 cells compared to the FLCN expressing ACHN and UOK257-F cells. Decreased BRCA1 A complex expression and disruption of DNA repair ability were detected in FLCN-deficient cells, suggesting that FLCN deficiency impaired BRCA1 A complex expression and sensitized cells to PARP inhibitor olaparib.

Conclusions

RCC cells deficient in FLCN are sensitive to olaparib treatment due to the impairment of BRCA1 A complex associated DNA repair ability. The results suggest that PARP inhibitor, such as olaparib, may be a potentially effective therapeutic approach for kidney tumors with deficiency of FLCN protein.

中文翻译：

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缺乏卵泡蛋白的肾癌细胞表现出BRCA1 A复合物表达受损和对PARP1抑制剂olaparib的敏感性

背景

Birt–Hogg–Dubé（BHD）疾病患者的卵泡蛋白（FLCN）缺乏可能导致肾细胞癌（RCC）。在这项研究中，我们调查了PARP抑制剂olaparib对FLCN缺乏的RCC细胞的细胞毒性，以及FLCN和BRCA1 A复合物调节的DNA修复途径之间的相互作用。

方法与材料

在这项研究中使用了表达FLCN的细胞（ACHN和UOK257-F）和缺乏FLCN的细胞（ACHN-2和UOK257）。通过克隆形成测定和MTT测定来检测细胞活力。流式细胞仪和TUNEL法检测细胞凋亡。通过MDC测定，蛋白质印迹和透射电子显微镜测量细胞中的自噬。进行了共免疫沉淀，免疫荧光和蛋白质印迹实验，以确定FLCN蛋白与BRCA1A复合物之间的相互作用。的体内实验在异种移植模型中通过在裸鼠接种UOK 257执行。

结果

具有FLCN蛋白缺陷的RCC细胞比具有FLCN表达的细胞对olaparib治疗更敏感。与表达FLCN的ACHN和UOK257-F细胞相比，Olaparib处理导致FLCN缺陷的ACHN-2和UOK257细胞中更严重的自噬和凋亡。在FLCN缺陷型细胞中检测到BRCA1 A复合物表达降低，DNA修复能力下降，这表明FLCN缺乏症损害了BRCA1 A复合物表达，并使细胞对PARP抑制剂olaparib敏感。

结论

由于BRCA1 A复合物相关的DNA修复能力受损，FLCN缺乏的RCC细胞对olaparib治疗敏感。结果表明，PARP抑制剂（例如olaparib）可能是治疗FLCN蛋白缺乏的肾脏肿瘤的潜在有效治疗方法。