HIF1α和TGFβ通路互作在重离子杀灭肿瘤干细胞中的作用

Cancer incidence and mortality have been increasing in China, making cancer the leading cause of death. Recent research findings strongly support that cancer stem cells (CSCs) are the main source of tumor proliferation, metastasis, and local treatment failure. The radioresistance and stemness of CSCs are regulated by both hypoxia‑inducible factor 1α (HIF1α) and transforming growth factor‑β (TGF β) in tumor microenvironments. Additionally, hypoxia-induced radioresistance is generally acknowledged as a major limiting factor for tumor control with radiotherapy, and HIF1α is responsible for increased stemness of CSCs by activating epithelial-mesenchymal transition (EMT). HIF1α subunit can be stabilized by TGFβ in tumor microenvironments. TGFβ promotes both pro‑angiogenic and anti‑angiogenic effects so that the development of strategies directly targeting TGFβ is likely to be challenging. The lower incidence of local relapse of heavy ion radiotherapy (HIRT) than conventional therapy with X-rays implies the possibility that heavy ions are more efficient in killing CSCs. Carbon ions with spread out Bragg peak display enhanced dose conformity and greater-than-one relative biological effectiveness (RBE), and rarely affected by the presence of oxygen. However, more research is needed to corroborate the findings and to confirm this hypothesis. More importantly, nothing is known about the optical radiation quality for efficient inactivation of CSCs, which is crucial for the optimization of HIRT treatment planning. In this project, we will obtain CSC cells and non-CSC cells by sorting lung cancer and ovarian cancer cells using stem cell surface markers, and expose them to carbon ions with various linear energy transfers (LETs) under either normoxic or hypoxic conditions. Cellular survival levels will be measured with routine colony-forming assay. Optimal LET will be therefore assumed to effectively kill tumor cells, especially CSCs by analyzing both RBE-LET and the oxygen enhancement ratio (OER)-LET curves. In this project, we will investigate the role of the interaction of HIF1α and TGF β pathway in the radiosensitivity and stemness of CSCs irradiated by heavy ions. The targeted inhibition of downstream molecules in TGF β pathway and HIF1α may be more beneficial for radiotherapy. Our proposed study will provide important new insight on the role of heavy ions in radiotherapy of tumors with large hypoxic fractions and to elucidate the radiosensitivity of CSCs to heavy ion radiotherapy.

肿瘤是居民的三大死因之一。肿瘤干细胞是肿瘤转移以及手术、化疗和放疗后复发的根源。肿瘤微环境中乏氧诱导因子（Hypoxia induced factor 1α，HIF1α）和TGFβ不仅调控肿瘤干细胞特性，也增加肿瘤细胞的辐射抗性。X射线可以诱导转录因子HIF1α稳定以及肿瘤微环境中TGFβ上调，而HIF1α稳定和TGFβ上调不仅促进肿瘤干细胞的干性，还影响肿瘤细胞的辐射敏感性。因此，肿瘤干细胞固有的辐射抗性是影响肿瘤放疗效果的主要障碍。与X射线等传统放疗手段相比较，重离子将Bragg峰准确定位于肿瘤靶区，不仅具有独特的物理学特性，而且具有明显的生物学优势，即相对生物效应（Relative Biological Effectiveness，RBE）强，氧增强比（Oxygen Enhancement Ratio，OER）小，对乏氧细胞和处于不同周期时相的肿瘤细胞均有较强的杀伤作用，因而对肿瘤干细胞具有较强的致死效应。所以，高能量的重离子可以靶向乏氧区的肿瘤干细胞，并且克服肿瘤干细胞固有的辐射抵抗，有效杀死肿瘤干细胞，提高肿瘤治疗效果。但是，相关实验研究十分有限。因此，本项目以男性发病率和死亡率最高的肺癌、女性病死率最高的卵巢癌为研究对象，以流式细胞术分选的人肺癌和卵巢癌干细胞为实验模型，以非肿瘤干细胞为对照模型，分别在正常和乏氧两种环境下，使用不同传能线密度（Linear Energy Transfer, LET）的碳离子束照射两对细胞实验模型，分析RBE-LET和ORE-LET剂量曲线，揭示对肿瘤干细胞和非肿瘤干细胞相对生物学效应强、氧效应低的最佳LET碳离子。另外，通过分析影响肿瘤干细胞干性和辐射敏感性的HIF1α和TGF β信号通路的互作关系，探讨两者共同调控的下游靶分子在碳离子杀伤肿瘤干细胞中的作用，为进一步优化肿瘤放射治疗方案提供可靠的理论支撑和实验数据。

肿瘤干细胞是肿瘤转移以及手术、化疗和放疗后复发的根源。肿瘤微环境中乏氧诱导因子（Hypoxia induced factor 1alpha，HIF 1alpha）和TGF β不仅调控肿瘤干细胞特性，也增加肿瘤细胞的辐射抗性。与X、gamma射线等传统光子放疗手段相比较，重离子对乏氧细胞和处于不同周期时相的肿瘤细胞均有较强的杀伤作用，因而对肿瘤干细胞具有较强的致死效应。所以，高能量的重离子可以靶向乏氧区的肿瘤干细胞，克服肿瘤干细胞固有的辐射抵抗，有较高的相对生物学效应（Relative Biologic Effectiveness, RBE），提高肿瘤治疗效果。本项目分析人和小鼠肿瘤干细胞在不同传能线密度（Linear Energy Transfer, LET）碳离子束照射后RBE-LET曲线，发现重离子束在100 LET时对人肿瘤干细胞的相对生物学效应最强，而非肿瘤干细胞的RBE随着LET升高而增加；本课题高通量分析He和碳离子照射正常氧和乏氧的A549细胞转录组的差异，发现碳离子辐射有特殊的ceRNA环状况，为后续探讨碳离子治疗肿瘤的机制提供明确的研究方向。并且发现X射线照射后转录本LINC00167和TGF-β1表达水平显著上调，与血管生成密切相关，为质子或重离子放疗后优于光子治疗后肿瘤复发和转移提供分子机制层面的解释，有望用于临床光子放疗辅助治疗靶点，改善光子放疗后患者的预后；另一方面，本课题也发现活性维生素D不仅提高正常氧条件下肿瘤细胞对X射线的辐射敏感性，也提高乏氧肿瘤细胞的辐射敏感性，这与激活VDR/NAPDH/ROS/apoptosis通路，抑制肿瘤细胞干性和诱导衰老相关。由于国内在建的质子照射装置逐年增加，作为一个天然存在的营养素，维生素D是否可以提高肿瘤细胞对质子和重离子辐射的敏感性还不清楚。因此，需要加快维生素D是否增加肿瘤细胞对不同LET离子的辐射敏感性的研究，通过设计靶向肿瘤细胞维生素D的纳米材料，促进本项目的实验室研究成果转化。