Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) - Cancer Biology, Rua Dr Roberto Frias, Porto, Portugal.
Endocr Relat Cancer. 2011 Jul 1;18(4):401-12. doi: 10.1530/ERC-10-0258. Print 2011 Aug.
Multiple endocrine neoplasia type 2 and a subset of apparently sporadic medullary thyroid carcinoma (AS-MTC) are caused by germ line activating point mutations of the rearranged during transfection (RET) proto-oncogene. RET encodes a receptor with tyrosine kinase activity that targets several intracellular signaling cascades, such as RAS-RAF-ERK1/2, PIK3-AKT, and STAT transcription factors. The objective of this study was to assess the function of three germ line RET variants Arg886Trp, Ser649Leu, and Glu511Lys of undetermined pathogenic significance, which were found in three kindreds of isolated AS-MTC. For this purpose, we employed vectors expressing each of the RET variants and measured the number of NIH3T3 transformation foci and soft agar colonies, the degree of activation of known RET intracellular signaling targets (ERK1/2, STAT1, STAT3, and TCF4), and the extent of ERK1/2 inhibition on sorafenib treatment. We found that RET variants Arg886Trp and Glu511Lys have shown increased in vitro transforming potential in a glial-derived neurotrophic factor-dependent manner. In contrast, the Ser649Leu variant did not significantly increased the number of foci and agar colonies relative to wild-type RET (RET-WT). The variants Glu511Lys and Arg886Trp showed 10- and 12.5-fold ERK1/2 activation respectively, that was significantly higher than that observed for RET-WT (fivefold). Increased levels of STAT1 and TCF4 activation were only observed for RET Arg886Trp (2.5- and 3-fold versus 1.2- and 2-fold in RET-WT respectively). The three RET variants analyzed here were sensitive to treatment with sorafenib. In conclusion, our results allow to classify previously uncharacterized RET genotypes, which may be of use to define follow-up and therapeutic regimens.
多发性内分泌肿瘤 2 型和部分散发型甲状腺髓样癌（AS-MTC）是由嵌合基因（RET）原癌基因突变引起的。RET 编码一种具有酪氨酸激酶活性的受体，可靶向几种细胞内信号通路，如 RAS-RAF-ERK1/2、PIK3-AKT 和 STAT 转录因子。本研究的目的是评估三个胚系 RET 变体 Arg886Trp、Ser649Leu 和 Glu511Lys 的功能，这些变体在三个孤立性 AS-MTC 家族中被发现，其致病性意义尚未确定。为此，我们使用表达每个 RET 变体的载体，并测量 NIH3T3 转化灶和软琼脂集落的数量、已知 RET 细胞内信号靶标的激活程度（ERK1/2、STAT1、STAT3 和 TCF4）以及 ERK1/2 在 sorafenib 治疗中的抑制程度。我们发现 RET 变体 Arg886Trp 和 Glu511Lys 在胶质源性神经营养因子依赖性方式下表现出增强的体外转化潜能。相比之下，Ser649Leu 变体相对于野生型 RET（RET-WT）并没有显著增加焦点和琼脂集落的数量。变体 Glu511Lys 和 Arg886Trp 的 ERK1/2 激活分别增加了 10 倍和 12.5 倍，明显高于 RET-WT（5 倍）。仅观察到 RET Arg886Trp 的 STAT1 和 TCF4 激活水平增加（分别为 2.5-和 3 倍，而 RET-WT 为 1.2-和 2 倍）。分析的三种 RET 变体对 sorafenib 的治疗敏感。总之，我们的结果允许对以前未表征的 RET 基因型进行分类，这可能有助于确定随访和治疗方案。
