SIVA mutation, a candidate metastasis gene identified from clonally related bilateral breast cancers, promotes breast cancer cell aggressiveness in vitro and in vivo

Our purpose is to identify candidate genes involved in the early steps of breast cancer metastasis and examine their pro-invasive functions both in vitro and in vivo. A percentage of bilateral breast cancers were clonally related based on copy number variation profiling. Whole exome sequencing and comparative sequence analysis revealed that a limited number of somatic mutations were acquired in this “breast to breast” metastasis. These mutations might promote breast cancer distant spread. The pro-invasive functions of a candidate metastasis gene were assessed in vitro by its abilities to promote proliferation, migration and invasion and in vivo as tumor xenografts in immunocompromised mice or a syngeneic orthotopic mouse breast cancer model. RNAseq analysis was performed to probe the transcription programs modulated by this candidate metastasis gene. SIVA1-D160N was one somatic mutation acquired in the breast to breast metastasis. Over-expression of SIVA1-D160N promoted migration and invasion of human MB-MDA-231 breast cancer cells in vitro, consistent with a dominant negative interfering function. When introduced via tail vein injection, 231 cells over-expressing SIVA1-D160N displayed enhanced distant spread on IVIS imaging. Over-expression of SIVA1-D160N promoted anchorage independent growth of mouse 4T1 breast cancer cells in vitro. When introduced orthotopically via mammary fat pad injection in syngeneic Balb/c mice, over-expression of SIVA1-D160N in 4T1 cells increased mammary gland tumor growth as well as liver metastasis. We conclude clonally related bilateral breast cancers represent a novel system to investigate metastasis and revealed a role of SIVA1-D160N in breast cancer metastasis. Differential gene expression analysis and pathway enrichment analysis of OVCAR8, SKOV3, HCC1954, and MDA-MB-231 parental cell lines as compared to their SIVA1-WT or SIVA1-D160N transformed counterparts.

本研究旨在鉴定参与乳腺癌转移早期进程的候选基因，并在体外（in vitro）与体内（in vivo）环境中验证其促侵袭功能。基于拷贝数变异谱分析，部分双侧乳腺癌存在克隆相关性。全外显子测序（whole exome sequencing）与比较序列分析显示，在这种“乳腺-乳腺”转移事件中仅获得了少量体细胞突变，此类突变或可促进乳腺癌的远端转移。

候选转移基因的促侵袭功能，可通过其促进细胞增殖、迁移与侵袭的能力在体外验证，亦可通过免疫缺陷小鼠的肿瘤异种移植模型或同基因原位小鼠乳腺癌模型在体内验证。本研究通过RNA测序（RNAseq）分析，探究该候选转移基因所调控的转录程序。

SIVA1-D160N即为该“乳腺-乳腺”转移事件中获得的体细胞突变之一。体外实验中，SIVA1-D160N的过表达可促进人MB-MDA-231乳腺癌细胞的迁移与侵袭，这与其显性负向干扰功能相符。通过尾静脉注射移植后，过表达SIVA1-D160N的231细胞在活体成像（IVIS）下呈现出更强的远端转移能力。体外实验中，SIVA1-D160N的过表达可促进小鼠4T1乳腺癌细胞的非锚定依赖性生长。在同基因Balb/c小鼠中通过乳腺脂肪垫原位移植后，4T1细胞中SIVA1-D160N的过表达可促进乳腺肿瘤生长，并增加肝脏转移负荷。

本研究结论表明，克隆相关性双侧乳腺癌是研究肿瘤转移的新型模型，并揭示了SIVA1-D160N在乳腺癌转移中的作用。本研究对OVCAR8、SKOV3、HCC1954及MDA-MB-231亲本细胞系，与其转染SIVA1-WT或SIVA1-D160N的对应细胞株开展了差异基因表达分析与通路富集分析。