REDOX COORDINATED EXTRACELLULAR MATRIX REMODELING IN CANCER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 低氧微环境和炎症促进了癌症的进行性进展，这些条件协同选择肿瘤细胞中改变的生存信号。低氧可诱导细胞死亡，导致细胞外基质的炎症和/或重塑，特别是通过沉积和处理细胞外基质聚合物组分，如透明质酸(HA)和胶原。我们假设，缺氧通过改变肿瘤细胞的氧化还原状态，以一种相互依赖的方式调节HA和胶原代谢酶，这种氧化还原状态和基质重塑之间的联系最终决定了肿瘤细胞的侵袭性表型。在这个试点项目中，我们将用三个具体目标来检验我们的假设。 目的1：确定参与HA和胶原代谢的基因是否在低氧和炎症引起的氧化应激反应中受到协同调控。我们将在有无促炎细胞因子的常氧和低氧条件下培养人前列腺癌细胞。我们将量化特定的HA合成酶和加工酶的表达，以及Pro代谢所需的酶，Pro代谢是一种抗氧化剂，也是胶原的主要成分，在细胞培养和我们存档的HA富含和HA缺乏的小鼠前列腺癌中。唐纳德·贝克尔博士将与我们合作进行这些研究。 目的2：量化缺氧和炎症对透明质酸和胶原生物合成和周转的影响。我们将测定在常氧和低氧条件下生长的肿瘤细胞的HA积累量、透明质酸酶活性、胶原酶活性和胶原生成。透明质酸酶的功能将与Joseph Barycki博士合作进行表征。与克雷顿大学的理查德·霍尔沃斯博士合作，将通过双光子显微镜量化NAD+/NADH比率和活性氧物种(ROS)来评估细胞的氧化还原状态。 目的：研究缺氧和炎症条件下透明质酸和胶原代谢改变的肿瘤细胞的表型。我们将在低氧和常氧条件下，通过操纵基因表达来检测肿瘤细胞的增殖、凋亡、细胞黏附和运动能力。参与介导这些反应的特定信号通路将从涉及运动性的氧化还原敏感转录因子(CTBP和HIF-1α)开始进行研究。 定义低氧、基质周转和肿瘤细胞行为之间的串扰将阐明低氧在肿瘤进展过程中促进纤维化组织损伤的机制，这是一个重要的临床问题。将改变的肿瘤细胞植入小鼠前列腺，检查纤维化/缺氧区与HA和胶原沉积的共存情况，以及对转移频率的评分，将建立潜在的临床相关性。进一步描述介导细胞对基质重塑反应的低氧基因调控通路将填补我们在理解肿瘤发生和转移方面的缺失环节，可能产生新的治疗方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Agressive cancer progression is promoted by hypoxic microenvironment and inflammation, conditions that synergize to select for altered survival signals in the tumor cells. Hypoxia may induce cell death that leads to inflammation and/or remodeling of the extracellular matrix, specifically through deposition and processing of extracellular matrix polymer components such as hyaluronan (HA) and collagen. We hypothesize that hypoxia, by altering the redox status of the tumor cell, regulates HA and collagen metabolizing enzymes in an interdependent fashion, and that this link between redox status and matrix remodeling ultimately determines aggressive tumor cell phenotype. In this pilot project, we will test our hypothesis with three specific aims. Aim 1: Determine whether genes involved in HA and collagen metabolism are regulated coordinately in response to oxidative stress imposed by hypoxia and inflammation. We will culture human prostate tumor cells in normoxic and hypoxic conditions in the presence and absence of pro-inflammatory cytokines. We will quantify expression of specific HA synthetic and processing enzymes, as well as enzymes required for proline metabolism, an antioxidant and major component of collagen, in cell culture and in our archived HA-rich and HA-deficient mouse prostate tumors. Dr. DOnald Becker will collaborate with us for these studies. Aim 2: Quantify changes in biosynthesis and turnover of HA and collagen in response to hypoxia and inflammation. We will measure HA accumulation, hyaluronidase activity, collagenase activity and collagen production in tumor cells grown under normoxic and hypoxic conditions as above. Hyaluronidase enzyme functions will be characterized in collaboration with Dr. Joseph Barycki. Cellular redox status will be evaluated by two-photon microscopic quantification of NAD+/NADH ratios and reactive oxygen species (ROS) in collaboration with Dr. Richard Hallworth at Creighton University. Aim 3: Evaluate phenotype of tumor cells with altered HA and collagen metabolism in response to hypoxia and inflammation. We will measure cell proliferation, apoptosis, cell adhesion and motility of tumor cells with manipulated gene expression in hypoxic and normoxic conditions. Specific signaling pathways involved in mediating these responses will be investigated beginning with redox-sensitive transcription factors implicated in motility (CTBP and HIF-1alpha). Defining crosstalk between hypoxia,matrix turnover and tumor cell behavior will elucidate mechanisms by which hypoxia promotes fibrotic tissue damage during tumor progression, a significant clinical problem. Implanting altered tumor cells in mouse prostates and examining colocalization of fibrotic/hypoxic regions with HA and collagen deposition, as well as scoring metastatic frequency, will establish potential clinical relevance. Further delineation of hypoxic gene regulation pathways that mediate cell responses to matrix remodeling will fill in missing links in our understanding of tumorigenesis and metastasis, possibly yielding new therapeutics.