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

描述(申请人提供)：转移是导致癌症死亡的首要原因。在肿瘤发生的所有过程中，局部侵袭和转移的形成是临床上最相关的，但了解最少。肿瘤内缺氧，多见于 实体瘤，与转移和治疗失败的风险增加有关。癌细胞通过增加缺氧诱导因子(HIF-1和HIF-2)的活性来适应低氧微环境。必须确定驱动HIF调节的转移的机制，以便确定有效的治疗策略，并有可能阻止转移。我们的初步数据显示，在体内和体外，HIF在HIF-1？在癌细胞和成纤维细胞中的依赖方式，通过胶原蛋白、脯氨酸羟基酶和赖氨酸羟基酶的转录上调。我们还发现，胶原羟基酶对于乳腺癌细胞向小鼠肺和淋巴结的自发转移是必不可少的。另一方面，我们发现缺氧以非细胞外基质依赖的方式诱导整合素受体的表达。这些观察结果使我们提出了一个模型，在该模型中，HIF同时诱导ECM成分(配体)及其整合素(受体)的产生，以增强下游信号事件，从而协同促进转移。这项拟议的研究将通过产生生理性的ECM底物来测试这一模型，这些底物概括了体内ECM的组成。在目标1中，我们将检验HIF-1或HIF-2转录调控癌细胞中几个整合素亚基表达的假设。在指导的K99阶段，我们将确定缺氧条件下整合素基因表达的HIF依赖模式。在独立的R00阶段，HIF对整合素的调节机制将被确定。在目标2中，我们将使用新的生物物理测试方法，在3D培养模型系统中验证低氧诱导和HIF依赖的整合素表达导致ECM黏附、运动性、侵袭性和基质收缩增加的假设。在指导的K99阶段，将产生细胞系来抵消缺氧对整合素表达的影响，并在所述的分析中进行测试。在目标3中，我们将检验这样一个假设，即在低氧条件下产生的细胞外基质上培养乳腺癌细胞将增强下游整合素信号。在K99期，我们将确定整合素的表达是否通过与低氧细胞外基质的相互作用而增强。在R00阶段，我们将检验这一假设，即在低氧条件下诱导的一些整合素是HIF诱导转移所必需的。有了这些信息，在R00阶段，我们将使用动物模型系统地评估转移级联中的每一步，以确定哪些步骤需要乳腺癌细胞表达整合素(S)(目标4)。综上所述，我们希望这些数据将导致转移性乳腺癌治疗的新策略。约翰霍普金斯大学独特的环境具有许多优势，这些优势将有助于支持我的培训和研究计划，以及我未来的科学生涯。我的主要导师塞门扎博士和共同导师丹尼斯·维尔茨博士都是各自领域的领导者。他们的领导力，再加上密集的职业发展培训计划和K99奖项，将有助于我向一名成功的独立科学家过渡。

DESCRIPTION (provided by applicant): 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.