Key Laboratory of Carcinogenesis and Translational Research, Department of Head and Neck, Peking University Cancer Hospital and Institute, Beijing, 100142, P.R. China.
Department of Pathology, Peking University Cancer Hospital and Institute, Beijing, 100142, P.R. China.
J Clin Endocrinol Metab. 2022 Aug 18;107(9):2636-2643. doi: 10.1210/clinem/dgac352.
Medullary thyroid cancer (MTC) is usually caused by gain-of-function mutations in the proto-oncogene RET.
This study aimed to determine the underlying mechanism in a male patient diagnosed with MTC at age 51 years.
Genomic DNA extracted from leukocytes or tumor tissues of patients was used for next-generation sequencing (NGS)-panel sequencing and Sanger sequencing. Wild-type (WT) and p.C630 deletion RET were expressed in HEK 293T cells. Activation of phosphorylation of the crucial tyrosine-905 of RET and MAPK/ERK was analyzed by Western blotting. The effect of RET mutants on cell viability and colony formation ability was determined by CCK8 assay and a colony forming assay.
NGS-Panel sequencing revealed a 3-nucleotide/1-amino acid C630 in-frame deletion in exon 11 of RET (c.1887_1889delGTG p.C630del). In vitro expression showed that phosphorylation of the crucial tyrosine 905 was much stronger in the p.C630del RET mutant than in WT RET, indicating ligand-independent activation of the Ret protein tyrosine kinase. Furthermore, p.C630del RET mutant induced strong activation of the MAPK/ERK pathway. In addition, p.C630del RET mutant cells exhibited increased HEK 293T cell viability and colony formation compared with WT RET cells. Pralsetinib (BLU-667), a highly selective RET inhibitor, inhibited the viability of WT RET and p.C630del RET mutant-transfected HEK 293T cells (IC50s: 18.54 and 16.49 µM after treatment for 24 hours), followed by inhibition of the RET-induced MAPK/ERK pathway.
The finding in our patient with MTC was a 3-base-pair deletion in exon 11 of RET, a p.C630 deletion not previously reported. The p.C630del RET stimulates cell proliferation by increasing ligand-independent phosphorylation and activation of MAPK/ERK pathway, demonstrating the pathogenic nature of the mutation. We therefore recommend screening panel sequence of RET in MTC patients with indications of a genetic cause.
甲状腺髓样癌（MTC）通常由原癌基因 RET 的功能获得性突变引起。
本研究旨在确定一名 51 岁男性 MTC 患者的潜在发病机制。
使用患者白细胞或肿瘤组织提取的基因组 DNA 进行下一代测序（NGS）-panel 测序和 Sanger 测序。将野生型（WT）和 p.C630 缺失 RET 表达于 HEK 293T 细胞中。通过 Western blot 分析关键酪氨酸残基 905 的磷酸化 RET 和 MAPK/ERK 的激活情况。通过 CCK8 测定和集落形成测定确定 RET 突变对细胞活力和集落形成能力的影响。
NGS-Panel 测序显示 RET 第 11 外显子 3 个核苷酸/1 个氨基酸 C630 框内缺失（c.1887_1889delGTG p.C630del）。体外表达显示，p.C630del RET 突变体中关键酪氨酸 905 的磷酸化强度明显强于 WT RET，表明 Ret 蛋白酪氨酸激酶的配体非依赖性激活。此外，p.C630del RET 突变体诱导 MAPK/ERK 通路的强烈激活。此外，与 WT RET 细胞相比，p.C630del RET 突变体细胞表现出更高的 HEK 293T 细胞活力和集落形成。普拉替尼（BLU-667）是一种高度选择性的 RET 抑制剂，抑制 WT RET 和 p.C630del RET 突变体转染的 HEK 293T 细胞活力（处理 24 小时后的 IC50 分别为 18.54 和 16.49 µM），随后抑制 RET 诱导的 MAPK/ERK 通路。
我们在一名 MTC 患者中发现了 RET 第 11 外显子的 3 个碱基缺失，这是一种以前未报道过的 p.C630 缺失。p.C630del RET 通过增加配体非依赖性磷酸化和激活 MAPK/ERK 通路来刺激细胞增殖，证明了突变的致病性。因此，我们建议对有遗传原因迹象的 MTC 患者进行 RET 基因panel 测序筛查。
Zotero 插件