Mechanisms of Stromal Cell Activation by the Developing Tumor

肿瘤基质通过促进肿瘤的生长和从原发肿瘤向转移部位的迁移来促进肿瘤的发展，这一点已经得到了很好的证实。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，被称为癌活化成纤维细胞（CAFs）。CAFs促进肿瘤进展的机制才刚刚开始出现。CAFs已被证明表达高水平的蛋白水解酶，包括基质金属蛋白酶（MMPs）和组织蛋白酶（1-3），这可能刺激肿瘤的传播和生长。CAFs可分泌趋化因子如单核细胞趋化蛋白-1和细胞因子如白细胞介素-1。此外，CAFs分泌基质来源因子-1 (SDF-1)，介导骨髓源性内皮细胞募集，直接促进肿瘤增殖。我们建立了人转移性乳腺癌细胞（MCF10CA1a）和正常小鼠真皮成纤维细胞的共培养系统，以确定基质和肿瘤间室之间串扰的影响。建立了两种类型的同型培养，分别是成纤维细胞和乳腺癌细胞，并与两种细胞类型的共培养进行了比较。我们能够证明，共培养的培养基增加了MCF10CA1a细胞在体外的迁移和散射，而同型培养的培养基几乎没有影响。一篇题为“通过tgf - β介导的机制，短暂的肿瘤-成纤维细胞相互作用持续增加肿瘤细胞恶性”的论文已经提交。为了进一步研究人类癌症中基质细胞重编程的机制，我们还建立了来自人类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。本项目的标本由弗吉尼亚大学妇科肿瘤科的合作者提供。该项目的目标是通过比较基因表达谱，使用微阵列方法和蛋白质表达，在癌症和正常标本中使用质谱，来表征与正常成纤维细胞相比，CAFs受影响的途径。由于成纤维细胞重编程的机制尚不清楚，我们也开始研究microrna的差异表达在肿瘤激活基质细胞中的作用，并分析其在促进肿瘤生长和/或转移中的可能功能。我们收集了7对正常/肿瘤来源的成纤维细胞，并通过上皮细胞和成纤维细胞标记染色来确定细胞群的纯度。我们还完成了mRNA和microRNA分析。初步结果表明，与已发表的数据一致，子宫内膜CAFs表达更高水平的MMPs和许多其他蛋白水解酶。TGF β 2的表达也上调数倍。我们还观察到IGF2和Wnt 5A的激活。这两种生长因子都是高度致瘤性的。除了Wnt5A的激活外，我们还发现分泌的卷曲相关蛋白（sFRP4）的表达降低，这是一种Wnt通路的抑制剂。这些数据经实时PCR验证。我们还研究了cas中sFRP下调的可能机制，发现它被甲基化抑制，而甲基化可以通过5-氮胞苷治疗逆转。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，鉴定出几种候选细胞，目前正在进行验证。我们鉴定了子宫内膜癌cas的11个microRNA特征，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的直接靶点。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。我们也在研究mir 148，并计划研究介导SAT B2的途径。

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.