核心蛋白聚糖（decorin）缺失的肿瘤微环境与结直肠癌发生和转移机制研究

Tumor microenvironment plays crucial roles in the development and metastasis of malignant tumors. Cancer progression and metastasis are the result of reciprocal interactions between cancer cells and their surrounding microenvironmental constituentsts. The tumor microenvironment encompasses cancer associated fibroblasts, inflammatory cells, angiogenesis and remodeling of extracellular matrix. Decorin is one of the main components of the extracellular matrix. We previously reported that in decorin-null mice there is a concurrent downregulation of E-cadherin and upregulation of β-catenin signaling. Both β-catenin and E-cadherin are hallmarks of epithelial-mesenchymal transition (EMT). E-cadherin loss promotes metastasis by disaggregating cancer cells from another, activates specific downstream signal transduction pathways and causes EMT, which facilitates metastasis. The focus of this proposal is the study of the role of decorin-deficient tumour microenvironment in colorectal cancer progression and metastasis. Specifically, we will investigate the cross-talk between decorin and EMT in colorectal cancer formation, invasion and metastasis. The result from this study would shed light on the colorectal cancer treatment using decorin targeting on the tumor microenvironment. We plan to: Aim 1: Investigate the effects of a decorin-deficient microenvironment on colorectal cancer formation and invasion in mice induced by azoxymethane (AOM). Aim 2: Study the anti-oncogenic properties of recombinant decorin protein core with or without the addition of decitabine in AOM induced decroin knockout colorectal cancer mice model. Aim 3: Investigate how a decorin-null microenvironment in host mice would affect colorectal cancer progression, metastasis, and how exogenous decorin would influence the tumor outcome. Aim 4: Utilize an in vitro co-culture syste of tumor fibroblasts and epithelial cells to discern the mechanism of cross-talk between decorin and EMT.

肿瘤微环境在恶性肿瘤的发展和转移中起着至关重要的作用。肿瘤的进展和转移是癌细胞与周围环境之间的相互作用的结果。肿瘤微环境由肿瘤相关的成纤维细胞、细胞外基质等成分组成。核心蛋白聚糖是细胞外基质的主要成分之一。 在前期的研究中E-cadherin蛋白下调和β-catenin蛋白的上调在核心蛋白聚糖缺陷小鼠被发现。β-catenin和E-cadherin这两种蛋白是上皮-间质转化（EMT ）的标志性分子。提示，核心蛋白聚糖与EMT之间有着密切的关系，本研究应用现代分子生物学，细胞生物学手段，结合偶氮甲烷AOM诱导的DCN野生型及基因缺失型的结直肠癌小鼠模型，研究DCN与上皮间质转换之间的相互作用在结直肠肿瘤发生、发展和转移过程中的作用，及核心蛋白聚糖对结直肠癌小鼠的治疗作用，研究成果为阐明结直肠癌的发病机制，也为将核心蛋白聚糖进一步开发为针对肿瘤微环境的新型防治结直肠癌生物制剂提供理论依据。

肿瘤的进展和转移是癌细胞与周围环境之间的相互作用的结果。肿瘤微环境由肿瘤相关的成纤维细胞、细胞外基质等成分组成。核心蛋白聚糖是细胞外基质的主要成分之一。本研究应用现代分子生物学，细胞生物学手段，结合偶氮甲烷AOM诱导的DCN野生型及基因缺失型的结直肠癌小鼠模型，研究DCN与上皮间质转换之间的相互作用在结直肠肿瘤发生、发展和转移过程中的作用，及核心蛋白聚糖对结直肠癌小鼠的治疗作用，研究结果发现：.与野生型小鼠相比，DCN的缺失的肿瘤微环境，会促进上皮间质转化的EMT过程。DCN基因敲除型小鼠的肠上皮细胞中MMP2的表达增加，上皮标志物E-cadherin的表达下降，同时间质标志物（N-cadherin、Vimentin、α-SMA）、EMT蛋白标志物分子（Snail、Slug、Twist）的表达均增加。DCN缺失之后还会上调COX-2和ICAM-1的表达，使其随着结直肠肿瘤浸润转移的发展而增加。 体外细胞水平，DCN重组蛋白和Celecoxib二者联合处理之后均能显著降低SW480细胞的增殖能力、抑制划痕愈合以及单个细胞集落形成能力。体内动物水平，DCN联合Celecoxib增强对AOM/DSSDCN基因敲除型结肠癌小鼠结直肠癌的侵袭与转移的抑制作用。其分子机制与EMT相关基因及MMP2的表达降低， E-cadherin的表达增加相关。DCN联合Celecoxib可显著降低小鼠体内Cox-2、PGE2的mRNA表达水平。.通过构建结肠癌细胞LOVO和结肠成纤维细胞CCD-18CO的共培养模型，从外源性和内源性在肿瘤微环境中过表达DCN，划痕实验及trans-well小室实验研究结果表明，DCN的过表达抑制结肠癌细胞的侵袭和转移。基质金属蛋白酶MMP2、MMP9的表达量下降，此外E-cadherin的表达量增加，间质标志物（Vimentin、α-SMA）、EMT蛋白标志物（Snail、Slug）、β-catenin及CDK4的表达量均下降。说明DCN在微环境中通过抑制结肠癌细胞的增殖侵袭和转移，来抑制其上皮间质转化的EMT过程。同时研究发现核心蛋白聚糖与活化素C存在之间的交互作用，对活化素C的负向调控作用也参与核心蛋白聚糖对结直肠肿瘤细胞浸润和转移的抑制作用。上述研究成果为阐明结直肠癌的发病机制，及核心蛋白聚糖开发为针对肿瘤微环境的新型防治结直肠癌生物制剂奠定基础。