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。几项研究表明，乳腺癌中氧响应性缺氧诱导因子（HIF-1a）蛋白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，肿瘤的缺氧区域是对放疗和化疗具有抗性的肿瘤细胞的主要来源。也有人提出缺氧刺激正常和癌症干细胞（CSC）的扩增。然而，HIF-1a对乳腺癌进展和转移与CSC的关系的具体贡献仍然不确定。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。将要检验的具体假设是，HIF-1a通过与Wnt/β-连环蛋白信号通路相互作用促进CSC更新、扩增和肿瘤起始能力，这增强了HIF-1a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更新和肿瘤起始活性，以及低氧暴露是否增强CSC活性。使用源自乳腺癌的MMTV-PyMT小鼠模型的新型HIF-1a野生型（WT）和无效（KO）肿瘤细胞，我们将通过比较肿瘤球形成效率和WT或KO细胞中肿瘤起始细胞的频率（针对表征鼠乳腺CSC的细胞表面标志物进行分选）来解决缺氧是否通过HIF-1a起作用以促进乳腺CSC更新。2)确定HIF-1a是否与Wnt/β-catenin信号通路相互作用以调节乳腺CSC行为。我们将决定，通过使用TS和有限稀释移植测定，如果HIF-1 α和<$-连环蛋白物理相互作用，并且如果CSC活性的Wnt/<$-连环蛋白调节是HIF-1 α依赖性的，则β-连环蛋白活性对于PyMT肿瘤细胞中的CSC活性是必需的。3)研究HIF-1a调节的CSC群体是否富集转移，并研究HIF-1a如何促进定植和转移。将使用实验转移的心内注射模型测试在目标1中发现受HIF-1a调节的CSC的流式分选亚群与亲本细胞相比的富集转移潜力。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。如果我们的假设，即HIF-1a通过与Wnt/<$-连环蛋白的相互作用增强CSC活性来促进乳腺肿瘤生长和转移得到证实，那么这可能对治疗模式产生重大影响，并表明可能进一步改进治疗方法，通过抗HIF和抗<$-连环蛋白治疗的组合来特异性根除乳腺CSC群体。此外，由于肿瘤起始小生境可能存在于缺氧微环境中，因此也可能利用抗HIF疗法与其他辅助疗法组合来预防转移性扩散，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.