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

描述（由申请人提供）：肿瘤生理学的一个关键方面是微环境中的氧气感觉。多项研究表明，氧反应性低氧诱导因子（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率降低相关。此外，肿瘤的缺氧区是肿瘤细胞对放疗和化疗耐药的重要来源。也有人提出，缺氧刺激正常和癌症干细胞（CSCs）的扩张。然而，HIF-1a在乳腺癌进展和转移中与CSCs的关系的具体贡献仍不明确。我们的长期目标是阐明缺氧如何通过HIF-1a调节乳腺CSC/祖细胞群体。具体的假设是HIF-1a通过与Wnt/¿-catenin信号通路的相互作用促进CSC的更新、扩展和肿瘤启动能力，这增强了HIF-1a的转录反应，导致原发性肿瘤生长和转移潜力增加。这将在三个具体目标中得到检验。1)确定HIF-1a是否促进乳腺CSC更新和肿瘤启动活性，以及低氧暴露是否增强CSC活性。利用MMTV-PyMT小鼠乳腺癌模型中衍生的新型HIF-1a野生型（WT）和非野生型（KO）肿瘤细胞，我们将通过比较WT或KO细胞中肿瘤球形成效率和肿瘤启动细胞的频率来研究缺氧是否通过HIF-1a促进乳腺CSC更新，这些细胞被分类为表征小鼠乳腺CSC的细胞表面标记物。2)确定HIF-1a是否与Wnt/¿-catenin信号通路相互作用调节乳腺CSC行为。我们将通过TS和限制稀释移植试验确定-catenin活性是否对PyMT肿瘤细胞中的CSC活性是必要的，HIF-1a和-catenin是否物理相互作用，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群体是否富集转移，以及HIF-1a如何促进定植和转移。在Aim 1中发现由HIF-1a调节的流式分类的CSCs亚群，将使用实验性转移的心内注射模型，与亲本细胞相比，测试其富集的转移潜力。慢病毒转导将用于确定HIF-1a调节的靶基因对这些过程的贡献。如果我们关于HIF-1a通过与Wnt/ -catenin相互作用增强CSC活性促进乳腺肿瘤生长和转移的假设得到证实，那么这可能对治疗模式产生重大影响，并表明有可能通过抗hif和抗-catenin疗法的结合进一步改进治疗方法，以特异性根除乳腺CSC群体。此外，由于肿瘤启动生态位可能存在于缺氧微环境中，因此也有可能将抗hif疗法与其他辅助疗法联合使用，以防止转移性扩散，或者在未来用于化学预防。

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.