E-Cadherin Destabilization Accounts for the Pathogenicity of Missense Mutations in Hereditary Diffuse Gastric Cancer

E-cadherin is critical for the maintenance of tissue architecture due to its role in cell-cell adhesion. E-cadherin mutations are the genetic cause of Hereditary Diffuse Gastric Cancer (HDGC) and missense mutations represent a clinical burden, due to the uncertainty of their pathogenic role. In vitro and in vivo, most mutations lead to loss-of-function, although the causal factor is unknown for the majority. We hypothesized that destabilization could account for the pathogenicity of E-cadherin missense mutations in HDGC, and tested our hypothesis using in silico and in vitro tools. FoldX algorithm was used to calculate the impact of each mutation in E-cadherin native-state stability, and the analysis was complemented with evolutionary conservation, by SIFT. Interestingly, HDGC patients harbouring germline E-cadherin destabilizing mutants present a younger age at diagnosis or death, suggesting that the loss of native-state stability of E-cadherin accounts for the disease phenotype. To elucidate the biological relevance of E-cadherin destabilization in HDGC, we investigated a group of newly identified HDGC-associated mutations (E185V, S232C and L583R), of which L583R is predicted to be destabilizing. We show that this mutation is not functional in vitro, exhibits shorter half-life and is unable to mature, due to premature proteasome-dependent degradation, a phenotype reverted by stabilization with the artificial mutation L583I (structurally tolerated). Herein we report E-cadherin structural models suitable to predict the impact of the majority of cancer-associated missense mutations and we show that E-cadherin destabilization leads to loss-of-function in vitro and increased pathogenicity in vivo.

E-钙粘蛋白（E-cadherin）因其在细胞间黏附过程中的核心作用，对维持组织架构至关重要。E-钙粘蛋白突变是遗传性弥漫型胃癌（Hereditary Diffuse Gastric Cancer, HDGC）的遗传学病因，而错义突变因其致病功能尚不明确，成为临床诊疗的一大负担。在体外（in vitro）与体内（in vivo）实验中，多数突变会导致蛋白功能丧失，但绝大多数突变的具体致病机制仍未明确。我们提出假说，认为E-钙粘蛋白的天然态稳定性降低可解释HDGC相关错义突变的致病性，并通过计算机模拟（in silico）与体外实验工具验证了该假说。本研究采用FoldX算法（FoldX algorithm）计算每一种突变对E-钙粘蛋白天然态稳定性的影响，并通过SIFT工具（SIFT）补充分析进化保守性特征。有趣的是，携带生殖系E-钙粘蛋白稳定性降低突变的HDGC患者，其确诊或死亡年龄更小，这提示E-钙粘蛋白天然态稳定性的丧失可解释该疾病的表型特征。为阐明E-钙粘蛋白稳定性降低在HDGC中的生物学意义，我们研究了一组新发现的HDGC相关突变（E185V、S232C与L583R），其中L583R被预测可降低蛋白稳定性。实验结果表明，该突变在体外无生物学活性，蛋白半衰期更短且无法完成成熟加工，这是由于其发生了蛋白酶体依赖性的过早降解；而通过人工突变L583I（结构上可耐受的突变）恢复蛋白稳定性后，该表型得以逆转。本研究报道了可用于预测绝大多数癌症相关错义突变影响的E-钙粘蛋白结构模型，并证实E-钙粘蛋白稳定性降低会在体外导致蛋白功能丧失，在体内提升疾病致病性。