The role of the hypoxic ECM on integrin-induced breast cancer metastasis

项目摘要 转移是癌症死亡的主要原因。在肿瘤发生的所有过程中， 局部浸润和转移的形成是最临床相关的，但了解最少。 在大多数实体瘤中发现的瘤内缺氧与以下风险增加相关： 转移和治疗失败。癌细胞适应缺氧微环境， 缺氧诱导因子（HIF-1和HIF-2）的活性。驱动HIF的机制- 必须确定受调节的转移，以确定有效的治疗策略， 可能阻断转移。我们的初步数据表明，HIF表达促进胶原蛋白 在癌细胞和成纤维细胞中， 胶原蛋白脯氨酰和赖氨酰羟化酶的转录上调。我们还发现胶原蛋白 羟化酶是乳腺癌细胞自发转移到肺所必需的 和小鼠的淋巴结。另一方面，我们发现缺氧诱导整合素的表达 受体以ECM独立的方式。这些观察使我们提出了一个模型， HIF同时诱导ECM组分（配体）及其整合素（受体）的产生 以增强协同增强转移的下游信号传导事件。拟议 研究将通过产生生理ECM底物来测试该模型， ECM在体内的组成。在目的1中，我们将检验HIF-1或HIF-2在转录水平上表达的假设， 调节癌细胞中几种整联蛋白亚基的表达。在辅导K99阶段， 我们将鉴定缺氧条件下整联蛋白基因表达的HIF依赖性模式。期间 在不依赖于R 00期的情况下，将确定整合素的HIF调节机制。在目标2中，我们 将检验缺氧诱导的和HIF依赖的整合素表达引起缺氧的假设。 在3D培养模型系统中ECM粘附、运动、侵袭和基质收缩增加， 新的生物物理测定。在指导的K99阶段，将产生细胞系以抵消 缺氧对整联蛋白表达的影响，并在所述测定中进行测试。在目标3中，我们将测试 在低氧条件下产生的ECM上培养乳腺癌细胞的假设 增强下游整合素信号传导。在K99阶段，我们将确定整合素 通过与缺氧ECM的相互作用增强表达。在R 00阶段，我们将测试 假设在缺氧条件下诱导的一些整合素是HIF诱导的细胞凋亡所必需的， 转移有了这些信息，在R 00阶段，我们将系统地评估 使用动物模型确定哪些步骤需要整联蛋白的转移级联中的步骤 乳腺癌细胞的表达（目的4）。总的来说，我们希望这些数据将导致新的 转移性乳腺癌的治疗策略。 约翰霍普金斯独特的环境有许多优势，将有助于支持我的 培训和研究计划以及我未来的科学事业。我的主要导师Semenza博士 共同导师Denis维尔茨博士是各自领域的领导者。他们的领导与一个 密集的职业发展培训计划和K99奖将有助于我过渡到一个 成功的独立科学家

Project Abstract Metastasis is the leading cause of cancer death. Of all the processes involved in tumorigenesis, local invasion and the formation of metastases are the most clinically relevant, but the least understood. Intratumoral hypoxia, found in the majority of solid tumors, is associated with an increased risk of metastasis and treatment failure. Cancer cells adapt to the hypoxic microenvironment by increasing the activity of the hypoxia-inducible factors (HIF-1 and HIF-2). The mechanisms that drive HIF- regulated metastasis must be determined in order to identify effective treatment strategies with the potential to block metastasis. Our preliminary data showed that HIF expression promotes collagen deposition in vivo and in vitro in a HIF-1¿ dependent manner in both cancer and fibroblast cells by the transcriptional upregulation of collagen prolyl and lysyl hydroxylases. We also showed that collagen hydroxylase enzymes were essential for the spontaneous metastasis of breast cancer cells to the lung and lymph nodes of mice. On the other hand, we found that hypoxia induced the expression of integrin receptors in an ECM-independent manner. These observations led us to propose a model in which HIFs simultaneously induce the production of ECM components (ligands) and their integrin (receptors) to potentiate downstream signaling events which synergistically enhance metastasis. The proposed research will test this model by generating physiological ECM substrates which recapitulate the composition of ECM in vivo. In aim 1, we will test the hypothesis that HIF-1 or HIF-2 transcriptionally regulates the expression of several integrin subunits in cancer cells. During the mentored K99 phase, we will identify the HIF-dependent pattern of integrin gene expression under hypoxia. During the independent R00 phase, the mechanism of HIF-regulation of integrins will be determined. In aim 2, we will test the hypothesis that hypoxia-induced and HIF-dependent integrin expression causes an increase in ECM adhesion, motility, invasion and matrix contraction in a 3D culture model system using novel biophysical assays. During the mentored K99 phase, cell lines will be generated to counteract the effect of hypoxia on integrin expression and tested in the assays described. In aim 3, we will test the hypothesis that culturing breast cancer cells on ECM produced under hypoxic conditions will potentiate downstream integrin signaling. In the K99 phase, we will determine whether integrin expression is potentiated by interactions with a hypoxic ECM. In the R00 phase, we will test the hypothesis that some of the integrins induced under hypoxic conditions are required for HIF-induced metastasis. With this information in hand, during the R00 phase, we will systematically evaluate each step in the metastatic cascade using animal models to determine which steps require integrin(s) expression by breast cancer cells (aim 4). Taken together, we hope this data will lead to novel strategies for the treatment of metastatic breast cancer. The unique environment at Johns Hopkins has many advantages that will serve to support my training and research plans as well as my future scientific career. My primary mentor, Dr. Semenza and co-mentor, Dr. Denis Wirtz, are leaders in their respective fields. Their leadership together with an intensive career development training plan and the K99 award will facilitate my transition to a successful independent scientist.