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。我们建立了人转移性乳腺癌细胞(MCF10CA1a)和正常小鼠皮肤成纤维细胞的共培养体系，以确定间质和肿瘤之间的串扰效应。建立了两种类型的同型培养，分别与成纤维细胞和乳腺癌细胞进行培养，并与两种细胞类型的共培养进行比较。结果表明，共培养条件下的MCF10CA1a细胞在体外迁移和分散增加，而同种培养条件下的MCF10CA1a细胞在体外迁移和分散的作用不明显。已经提交了一篇手稿，题为《短暂的肿瘤-成纤维细胞相互作用通过转化生长因子-β介导的机制持久地增加肿瘤细胞的恶性程度》。为了进一步研究人类肿瘤间质细胞重编程的机制，我们还建立了来源于人子宫内膜癌标本和患者匹配的正常子宫内膜组织的成纤维细胞系。该项目的标本由弗吉尼亚大学妇科肿瘤科的合作者捐赠。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。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。我们鉴定了子宫内膜癌CAF的11个microRNA信号，并表明miR-31抑制癌细胞的迁移和侵袭。我们发现SATB2染色质重塑蛋白是miR-31的直接靶标。对成纤维细胞中SATB2蛋白水平的调控表明其具有诱导细胞运动的功能。我们还在调查MIR148，并计划调查SAT B2的调节途径。

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. We established a coculture system with human metastatic breast cancer cells (MCF10CA1a) and normal murine dermal fibroblasts in order to determine the effects of crosstalk between the stromal and tumor compartments. Two types of homotypic cultures, with fibroblasts and breast cancer cells respectively, were established and compared to a coculture of both cell types. We were able to show that medium conditioned by cocultures of increased migration and scattering of MCF10CA1a cells in vitro, whereas medium conditioned by homotypic cultures had little effect. A manuscript, "Transient tumor-fibroblast interactions durably increase tumor cell malignancy by a TGF-beta mediated mechanism" has been submitted. To further investigate the mechanisms of stromal cell reprogramming in human cancers, we also established fibroblast cell lines derived from human endometrial cancer specimens and patient matched normal endometrial tissue. Specimens for this project were contributed by collaborators in 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. We also observed activation of IGF2 and Wnt 5A. Both of these growth factors are highly tumorigenic. In addition to 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. We identified an 11 microRNA signature of endometrial cancer CAFs and showed that miR-31 represses cancer cells migration and invasion. We identified SATB2 chromatin remodeling protein as a direct target of miR-31. Manipulation of SATB2 protein levels in fibroblasts suggest its function in inducing cell motility. We are also investigating mir 148 and plan to investigate the pathways by which SAT B2 is mediated.