Mechanisms of Stromal Cell Activation by the Developing Tumor

众所周知，肿瘤间质通过促进肿瘤的生长和从原发肿瘤向转移部位的转移来促进肿瘤的发展。基质反应细胞的一个组成部分是正常周围组织中的成纤维细胞，它们被肿瘤重新编程，被称为癌症激活的成纤维细胞(CAF)。CAF促进肿瘤进展的机制才刚刚开始出现。研究表明，CAF表达高水平的蛋白水解酶，包括基质金属蛋白酶(MMPs)和组织蛋白(1-3)，这可能刺激肿瘤的扩散和生长。CAF可分泌趋化因子，如单核细胞趋化蛋白-1和细胞因子，如白介素1。此外，CAF分泌基质衍生因子-1(SDF-1)，它介导骨髓来源的内皮细胞募集，并直接促进肿瘤增殖。为了探讨人类肿瘤间质细胞重编程的机制，我们建立了来源于人类子宫内膜癌标本和匹配的正常子宫内膜组织的成纤维细胞系。该项目是与弗吉尼亚大学妇科肿瘤科合作进行的。该项目的目标是通过比较癌症和正常标本中的基因表达谱、微阵列方法和蛋白质表达谱，与正常成纤维细胞相比，表征CAF中受影响的途径。由于成纤维细胞重编程的机制(S)尚不清楚，我们还着手研究microRNAs的差异表达在肿瘤间质细胞激活中的作用，并分析其在促进肿瘤生长和/或转移方面的可能功能。我们收集了7对正常/肿瘤来源的成纤维细胞，并用上皮标记物和成纤维细胞标记物染色来确定细胞群的纯度。我们还完成了信使核糖核酸和微核糖核酸的分析。初步结果表明，与已发表的数据一致，子宫内膜CAF表达更高水平的MMPs和其他一些蛋白水解酶。转化生长因子β2的表达也上调了数倍。此外，我们还观察到IGF2和WNT5A的激活。这两种生长因子都具有很强的致瘤性。除了Wnt5A的激活外，我们还发现Wnt途径的抑制物分泌型FrizzledRelated Protein(SFRP4)的表达减少。这些数据通过实时荧光定量聚合酶链式反应进行了验证。我们还研究了在CAF中SFRP下调的可能机制，发现它被甲基化抑制，而甲基化可以被5-氮杂胞苷处理逆转。对来自正常邻近组织的CAF和成纤维细胞差异表达的microRNA进行分析，结果发现了几个目前正在验证的候选基因。目前，我们正在为进一步验证结果做准备，方法是将microRNAs与我们微阵列签名中预测的目标基因(S)进行匹配。这一筛选使我们能够识别可能与肿瘤进展有关的几个假定靶点。其中一对是miR31(在CAF中减少)和SATB2基因(在CAF中增加)。我们发现，miR-31在来自癌症样本的成纤维细胞中的过表达下调了内源性SATB2的表达。当我们使用其他类型的成纤维细胞(正常皮肤或乳房成纤维细胞)时，也得到了类似的结果。

It is well established that the tumor stroma contributes to tumor development by promoting tumor growth and migration from the primary tumor to metastatic sites. One of the components of stromal responsive cells are fibroblasts in the normal surrounding tissue that are reprogrammed by the tumor and are referred to as cancer-activated fibroblasts (CAFs). The mechanisms whereby CAFs promote tumor progression are only beginning to emerge. CAFs have been shown to express high levels of proteolytic enzymes, including matrix metalloproteinases (MMPs) and cathepsins (1-3), which may stimulate tumor dissemination as well as growth. CAFs may secrete chemokines such as monocyte chemotactic protein-1 and cytokines such as interleukin-1. In addition, CAFs secrete stromal-derived factor-1 (SDF-1) which mediates bone marrow-derived endothelial cell recruitment and directly increases tumor proliferation. To investigate the mechanisms of stromal cell reprogramming in human cancers, we established fibroblast cell lines derived from human endometrial cancer specimens and matched normal endometrial tissue. This project was performed in collaboration with the Division of Gynecologic Oncology, University of Virginia. The goal of the project is to characterize the pathways affected in CAFs compared to normal fibroblasts by comparing gene expression profiles, using a microarray approach and protein expression, using mass spectroscopy in cancer and normal specimens. Since the mechanism(s) of fibroblast reprogramming are unknown, we also set out to investigate the role of differential expression of microRNAs in stromal cell activation by tumors and to analyze its possible function in promoting tumor growth and/or metastasis. We accumulated 7 pairs of normal/tumor-derived fibroblasts and determined the purity of cell population by staining with epithelial and fibroblasts markers. We also completed mRNA and microRNA profiling. The preliminary results show that, in agreement with published data, endometrial CAFs express higher levels of MMPs and a number of other proteolytic enzymes. The expression of TGF beta2 is also upregulated several fold. In addition we observed activation of IGF2 and Wnt 5A. Both of these growth factors are highly tumorigenic. Besides the activation of Wnt5A, we found decreased expression of secreted Frizzled related protein (sFRP4), an inhibitor of the Wnt pathway. These data were validated by real-time PCR. We also studied the possible mechanisms of sFRP downregulation in CAFs and found that it is suppressed by methylation that can be reversed by 5-azacytidine treatment. Profiling of microRNA differentially expressed by CAFs and fibroblasts from normal adjacent tissue resulted in identification of several candidates that are now being validated. At present we are preparing for the further validation of the results by matching the microRNAs with their predicted target gene(s) present in our microarray signature. This screen allowed us to identify several putative targets that maybe involved in tumor progression. One such pair is miR31 (reduced in CAFs) and the SATB2 gene (increased in CAFs). We showed that overexpression of miR-31 in fibroblasts derived from cancer samples downregulated expression of endogenous SATB2 mRNA. Similar results were obtained when we used other types of fibroblasts (normal skin or breast fibroblasts).