The Role of HIF-1alpha in Breast Cancer Stem Cell Activity

描述(申请人提供)：肿瘤生理学的一个关键方面是微环境中的氧气感觉。一些研究表明，氧反应低氧诱导因子(HIF-1a)蛋白在乳腺癌中的过度表达与预后不良、转移风险增加和生存期降低相关。此外，肿瘤的缺氧区是抵抗放射和化疗的肿瘤细胞的重要来源。也有人提出，低氧刺激正常干细胞和癌症干细胞(CSCs)的扩张。然而，HIF-1a在乳腺癌进展和转移中的具体作用与CSCs的关系仍不明确。我们的长期目标是阐明低氧是如何通过HIF-1a调节乳腺CSC/祖细胞数量的。需要检验的具体假设是，HIF-1a通过与Wnt/β-catenin信号通路相互作用，促进CSC的更新、扩张和肿瘤启动能力，从而增强HIF-1a的转录反应，从而增加原发肿瘤的生长和转移潜能。这将在三个具体目标上进行测试。1)确定HIF-1a是否促进乳腺CSC的更新和肿瘤的启动活性，以及低氧暴露是否增强CSC的活性。使用来自MMTV-PYMT小鼠乳腺癌模型的新型HIF-1a野生型(WT)和空(KO)肿瘤细胞，我们将通过比较肿瘤球体形成效率和WT或KO细胞中肿瘤起始细胞的频率来研究缺氧是否通过HIF-1a促进乳腺CSC更新，WT或KO细胞被分选为表征小鼠乳腺CSCs的细胞表面标志。2)确定HIF-1a是否与Wnt/β-catenin信号通路相互作用来调节乳腺CSC的行为。我们将通过TS和有限稀释移植试验来确定？-catenin活性是否是PYMT肿瘤细胞CSC活性所必需的，HIF-1a和？-catenin是否存在物理相互作用，以及Wnt/？-catenin对CSC活性的调节是否依赖于HIF-1a。3)研究HIF-1a调控的CSC是否富含转移能力，以及HIF-1a如何促进CSC的侵袭和转移。在Aim 1中发现受HIF-1a调控的CSCs的流动分选亚群将使用实验性转移的心内注射模型，与亲本细胞相比，测试其丰富的转移潜力。慢病毒转导将用于确定HIF-1a调控的靶基因在这些过程中的作用。如果我们的假设得到证实，即HIF-1a通过与Wnt/？-catenin的相互作用增强CSC的活性，从而促进乳腺肿瘤的生长和转移，那么这可能会对治疗模式产生重大影响，并可能进一步改进治疗，通过联合抗HIF和抗-catenin治疗来特异性地根除乳腺CSC群体。此外，由于启动肿瘤的生态位可能存在于低氧微环境中，因此也有可能将抗HIF疗法与其他辅助疗法结合使用，以防止转移扩散，或在未来用于化学预防。 与公共健康相关：几条证据表明，HIF-1a蛋白有助于肿瘤细胞在乳房生长并转移到身体中较远的位置(转移)，最终降低患者的存活率。我们现在寻求解决的问题是，HIF-1a促进这些过程的关键方式是否通过调节乳腺癌中一小部分细胞的行为，这些细胞被认为具有增强的自我更新和肿瘤启动活性，即“癌症干细胞”。如果HIF-1a调节癌症干细胞的功能，那么针对HIF-1a途径进行药物设计将对患者有利，因为干扰低氧反应的主调节因子可能会扰乱肿瘤生长和侵袭所必需的多个下游过程，实际上可能优先杀死癌症干细胞。

DESCRIPTION (provided by applicant): A critical aspect of tumor physiology is the sensation of oxygen in the microenvironment. Several studies have demonstrated that over-expression of the oxygen-responsive Hypoxia-Inducible Factor (HIF-1a ) protein in breast cancer correlates with poor prognosis, increased risk of metastasis and decreased survival. Moreover, hypoxic regions of tumors are a prominent source of tumor cells that are resistant to radiation and chemotherapy. It has also been proposed that hypoxia stimulates expansion of normal and cancer stem cells (CSCs). Yet, the specific contribution of HIF-1a to breast cancer progression and metastasis in relationship to CSCs remains undefined. Our long-term goal is to elucidate how hypoxia, through HIF-1a , regulates breast CSC/progenitor populations. The specific hypothesis that will be tested is that HIF-1a promotes CSC renewal, expansion and tumor-initiating ability through interactions with the Wnt/¿-catenin signaling pathway, which potentiates the HIF-1a transcriptional response, resulting in increased primary tumor growth and metastatic potential. This will be tested in three specific aims. 1) To determine if HIF-1a promotes breast CSC renewal and tumor-initiating activity and if hypoxic exposure enhances CSC activity. Using novel HIF-1a wild type (WT) and null (KO) tumor cells derived from the MMTV-PyMT mouse model of breast cancer, we will address if hypoxia acts through HIF-1a to promote breast CSC renewal by comparing tumorsphere formation efficiencies and the frequency of tumor initiating cells in WT or KO cells that are sorted for cell surface markers that characterize murine mammary CSCs. 2) To determine if HIF-1a interacts with the Wnt/¿-catenin signaling pathway to regulate breast CSC behavior. We will determine if ¿-catenin activity is necessary for CSC activity in PyMT tumor cells, if HIF-1a and ¿-catenin physically interact and if Wnt/¿-catenin regulation of CSC activity is HIF-1a dependent by using TS and limiting dilution transplantation assays. 3) To investigate if HIF-1a -regulated CSC populations are enriched for metastasis and to investigate how HIF-1a promotes colonization and metastasis. Flow-sorted sub-populations of CSCs found to be regulated by HIF-1a in Aim 1 will be tested for enriched metastatic potential compared to parental cells using an intracardiac injection model of experimental metastases. Lentiviral transduction will be used to determine the contribution of HIF-1a regulated target genes to these processes. If our hypotheses that HIF-1a promotes breast tumor growth and metastasis through enhancing CSC activity via interactions with the Wnt/¿-catenin are confirmed, then this could have significant impact on treatment paradigms, and would suggest that it may be possible to further refine treatments to specifically eradicate the breast CSC population through a combination of anti-HIF and anti- ¿-catenin therapies. In addition, since the tumor-initiating niche may exist in a hypoxic microenvironment, it may also be possible to utilize anti-HIF therapies in combination with other adjuvant therapies to prevent metastatic spread or, in the future, for chemoprevention. PUBLIC HEALTH RELEVANCE: Several lines of evidence suggest that the HIF-1a protein helps tumor cells to grow in the breast and to travel to distant sites in the body (metastasis), ultimately reducing patient survival. We now seek to address whether a key way by which HIF-1a promotes these processes is through regulation of behavior of a small fraction of cells within breast cancers that are believed to have enhanced self-renewal and tumor-initiating activity, the "cancer stem cells". If HIF-1a regulates cancer stem cell function, then targeting the HIF-1a pathway for drug design would be beneficial to patients since interfering with a master regulator of the hypoxic response could disrupt multiple downstream processes essential to tumor growth and invasion, and may in fact, preferentially kill the cancer stem cells.