Mechanisms of Stromal Cell Activation by the Developing Tumor

已经确定，肿瘤间质通过以下方式促进肿瘤的发展： 促进肿瘤生长和从原发肿瘤向转移部位的迁移。之一 基质反应细胞的组分是正常周围组织中的成纤维细胞， 被肿瘤重编程，被称为癌症激活的成纤维细胞（CAF）。的 CAF促进肿瘤进展的机制才刚刚开始出现。CAF已经 显示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），其可刺激肿瘤扩散以及生长。CAFs 可以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分泌基质衍生因子-1（SDF-1），其介导骨 骨髓来源的内皮细胞募集，并直接增加肿瘤增殖。我们 建立了与人转移性乳腺癌细胞（MCF 10 CA 1a）的共培养体系， 正常鼠皮肤成纤维细胞，以确定 基质和肿瘤区室。两种类型的同型培养物，具有成纤维细胞和乳腺癌细胞。 分别建立癌细胞，并与两种细胞类型的共培养物进行比较。我们 能够证明，通过增加迁移和散射的共培养物调节的培养基 MCF 10 CA 1a细胞在体外条件培养液中， 效果一份手稿，“短暂的肿瘤-成纤维细胞相互作用持久地增加肿瘤细胞 通过TGF-β介导的机制引起的恶性肿瘤”的论文已经提交。进一步调查 为了研究人类癌症中基质细胞重编程的机制，我们还建立了成纤维细胞 来源于人子宫内膜癌标本和患者匹配的正常子宫内膜的细胞系 组织.该项目的标本由海洋科学司的合作者提供。 妇科肿瘤学，弗吉尼亚大学。该项目的目标是描述 通过比较基因表达，与正常成纤维细胞相比，CAFs中受影响的途径 谱，使用微阵列方法和蛋白质表达，使用质谱， 癌症和正常标本。由于成纤维细胞重编程的机制是未知的， 我们还着手研究microRNA的差异表达在间质细胞中的作用， 肿瘤细胞激活，并分析其在促进肿瘤生长中的可能功能 和/或转移。我们收集了7对正常/肿瘤来源的成纤维细胞，并确定了它们的细胞分化情况。 通过上皮和成纤维细胞标记物染色确定细胞群的纯度。我们也 完成mRNA和microRNA分析。初步结果显示，与 已发表的数据显示，子宫内膜CAF表达更高水平的MMPs和许多其他的 蛋白水解酶TGF β 2的表达也上调了数倍。我们也 观察到IGF 2和Wnt 5A的活化。这两种生长因子都具有高度致瘤性。 除了Wnt 5A的激活，我们发现分泌的Frizzled表达减少， 相关蛋白（sFRP 4），Wnt通路的抑制剂。这些数据得到了验证， 实时荧光定量PCR我们还研究了sFRP在CAFs中下调的可能机制， 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。 CAFs和正常人成纤维细胞差异表达microRNA的分析 组织的结果是确定了几个候选人，现在正在验证。我们 鉴定了子宫内膜癌CAFs的11个microRNA特征，并显示miR-31 抑制癌细胞的迁移和侵袭。我们发现SATB 2染色质重塑 蛋白质作为miR-31的直接靶点。成纤维细胞中SATB 2蛋白水平的操纵 提示其在诱导细胞运动中的功能。我们也在调查和平号148， 研究SAT B2介导的途径。

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.