肿瘤胞外囊泡通过微小RNA刺激成纤维细胞向利于肿瘤转移方向分化的机制研究

Every year, thousands of women around the world die of breast cancer. About 90% of breast cancer deaths are due to metastasis, a stage at which cancers have spread to distant sites and treatments become ineffective. The development of effective therapies for cancer metastasis is an urgent unmet clinical need. This requires a thorough understanding of the evolving interaction between tumor cells and their environment at different stages of metastasis. During metastasis, tumor cells release large quantities of small vesicles that can deliver proteins and nucleic acids to other cells. Recent studies demonstrated that these vesicles contain large amounts of microRNAs (miRNAs), small RNAs that regulate gene expression. We illustrated that a group of miRNAs in tumor vesicles, the miR-200 family, can propagate metastatic capability in tumor cells. Our data reveal that miR-200 miRNAs are secreted by metastatic breast cancer cells and enriched in the circulation of mice bearing human metastatic tumors. Moreover, uptake of miR-200s by poorly metastatic tumor cells enhanced colonization of these cells in the lung. However, it remains unclear whether tumor-derived miR-200s also influence other cells in the tumor microenvironment. Do healthy cells take up miR-200s, and can that alter the metastatic progression of disease?.To identify host cells that naturally take up vesicles from tumor cells, we created breast cancer cells that secrete fluorescent vesicles. These fluorescent vesicles were taken up the most by fibroblasts in the tumors (unpublished data). In tumors, fibroblasts and endothelial cells often give rise to cancer-associated fibroblasts (CAFs) that are similar to myofibroblasts in phenotype and gene expression. CAFs play a key role in supporting tumor growth and metastasis. We hypothesize that miR-200s are transferred from tumor cells to fibroblasts via small vesicles and promote the conversion of fibroblasts into CAFs. .We propose to quantify the uptake of tumor-secreted miR-200s by fibroblasts in primary breast tumors and in the lung, a major metastatic site for breast cancer cells (Aim 1). Subsequently, we will examine the influence of miR-200s uptake on the differentiation of fibroblasts (Aim 2). .The outcome of this study will illustrate the novel contribution of secreted miR-200s to the crosstalk between cancer cells and stromal cells during metastasis. Such a mechanism could suggest therapeutic approaches to targeting miR-200s in tumor vesicles for the treatment of breast cancer metastasis.

肿瘤细胞释放的大量胞外囊泡中含有大量能影响基因表达的微小RNA(miRNAs)和小RNA。我们证明了其中的一个miRNA家族,miR-200，能诱导弱转移肿瘤细胞发生癌症转移。但是，胞外囊泡是否影响肿瘤微环境，使之利于癌症转移？ 为了鉴别哪些肿瘤基质细胞吸收肿瘤细胞释放的胞外囊泡，我们建立能释放荧光胞外囊泡的乳腺癌肿瘤细胞。这些荧光囊泡大多被成纤维细胞吸收（未发表）。在肿瘤中，成纤维细胞和上皮细胞通常成为癌症相关成纤维细胞（cancer-associated fibroblasts (CAFs)）。CAFs在形态上和基因表达上和肌成纤维细胞类似， 并在支持肿瘤生长和转移上有重要作用。我们推测胞外囊泡介导的miR-200自肿瘤细胞到成纤维细胞的转移促进了成纤维细胞向CAFs的转变。我们将定量检测在肿瘤部位和主要的肿瘤转移部位的成纤维细胞吸收源自初代肿瘤的miR-200。随之，我们将研究吸收的miR-200在成纤维细胞分化中的功能。这项研究将阐明在癌症转移中，分泌型miR-200在肿瘤细胞和基质细胞的交流中的重要贡献。这个机制预示了拮抗胞外小体中的miR-200是潜在的治疗乳腺癌转移的疗法

90%乳腺癌女性患者是由于癌症转移导致死亡，因此亟待开发高效治疗癌症转移疗法。以往研究表明，肿瘤细胞与其微环境的相互作用在肿瘤转移过程中起到重要作用，肿瘤细胞可释放大量细胞外囊泡（EVs）介导其与肿瘤微环境的相互作用。本项目已证实乳腺肿瘤和肺转移灶中的基质细胞摄取肿瘤细胞来源的细胞外囊泡；RNA测序表明，在转移性乳腺癌细胞分泌的细胞外囊泡中miR-125b显著高表达；同时，miR-125b被释放在肿瘤细胞外囊泡中并可被成纤维细胞摄取；在同基因4TO7肿瘤中，4T1 EVs 处理可诱导成纤维细胞激活，阻断4T1 EVs中的miR-125b可逆转上述激活作用；相关机制研究表明，miR-125b通过抑制Tp53inp1以激活小鼠成纤维细胞；人源乳腺癌细胞分泌miR-125b至细胞外囊泡并将其转移至肿瘤相关成纤维细胞，并通过抑制TP53INP1和TP53以促进其活化；小鼠和人成纤维细胞中过表达miR-125b可诱导出与构建敲除miR-125b靶mRNAs相似的活化表型。以上体内外实验证实，转移性乳腺癌细胞胞外囊泡可通过释放miR-125b，并通过抑制TP53INP1和TP53诱导成纤维细胞活化，并促进其发展为癌症相关的成纤维细胞。本研究验证了肿瘤胞外囊泡对肿瘤微环境中成纤维细胞分化的作用，为乳腺癌转移机制及其治疗提供了新的思路。