Investigation of breast cancer-associated mutations in RND1 gene

We identified the Rho family GTPase Rnd1 as a candidate breast tumor suppressor by using bioinformatics analysis in conjunction with iRNA silencing. Upon silencing of Rnd1, normal mammary epithelial cells underwent a complete EMT but eventually became senescent, suggesting that they had experienced an oncogenic insult. Expression of c-Myc rescued the Rnd1-depleted cells from senescence by suppressing p27Kip and it enabled their neoplastic conversion. The resulting cells were able to grow in soft agar and to form invasive, multi-acinar structures in 3D Matrigel. Silencing of Rnd1 promoted disruption of epithelial adhesion and polarity in xenograft models. In contrast, expression of Rnd1 inhibited tumor development. Mechanistic studies revealed that Rnd1 suppresses Ras signaling and prevents the EMT by activating the Ras-GAP domain of Plexin B1. Finally, analysis of clinical samples and cancer cell lines provided evidence that gene deletion, epigenetic silencing, and missense mutations contribute to inactivate RND1 in a subset of human breast cancers. These results indicate that Rnd1 promotes epithelial adhesion and polarity and suppresses oncogenesis by restraining unscheduled activation of Ras. genomic DNA from 96 breast cancer tissues and 8 normal mammary tissues were extracted and subjected to SOLID deep sequencing for exons of the RND1 gene to identify tumor-associated mutations with the amino acid exchanges There were two separate SOLID runs (0136 and 0270), on the same 96 tumors. The pools are indicated by "A1", "A2", "B1", etc.

本研究通过生物信息学分析结合RNA沉默（RNA silencing，原文标注iRNA）技术，鉴定出Rho家族GTP酶（Rho family GTPase）Rnd1为候选乳腺癌抑癌基因。敲低Rnd1后，正常乳腺上皮细胞会发生完全的上皮间质转化（Epithelial-Mesenchymal Transition, EMT），但最终进入衰老状态，提示细胞受到了致瘤性应激。过表达c-Myc可通过抑制p27Kip的表达，挽救Rnd1敲低细胞的衰老表型，并使其发生肿瘤性转化。所得细胞可在软琼脂中增殖，且能在三维基质胶（3D Matrigel）中形成侵袭性多腺泡结构。在异种移植模型（xenograft models）中，Rnd1沉默会促进上皮黏附与极性的破坏。与之相反，过表达Rnd1可抑制肿瘤发生。机制研究表明，Rnd1可通过激活Plexin B1的Ras-GAP结构域（Ras-GAP domain），抑制Ras信号通路（Ras signaling）并阻断上皮间质转化。最后，对临床样本与癌细胞系的分析证实，基因缺失、表观遗传沉默（epigenetic silencing）以及错义突变（missense mutations）会在部分人类乳腺癌中导致RND1基因失活。上述结果表明，Rnd1可通过抑制Ras的异常激活，维持上皮黏附与极性，并抑制肿瘤发生。本研究提取了96份乳腺癌组织与8份正常乳腺组织的基因组DNA，针对RND1基因的外显子（exons）区域进行SOLID深度测序（SOLID deep sequencing），以鉴定携带氨基酸替换的肿瘤相关突变。同一批96份肿瘤样本共开展了两轮独立的SOLID测序（编号0136与0270），测序文库以"A1"、"A2"、"B1"等标识区分。