Mechanisms of progression of vascular malformation to lymphangiosarcoma

摘要/摘要 内皮细胞是血管和淋巴管的重要组成部分，也是异常的内皮细胞。 由于各种信号通路的异常导致的增殖导致了广泛的血管异常 包括血管畸形和肿瘤。淋巴管畸形和淋巴管肉瘤分别为 起源于淋巴管内皮细胞的血管异常。虽然LM大部分仍是一种良性疾病，但有一小部分 LM患者进展为高度侵袭性和致命性LAS。虽然LM(如乳房慢性淋巴水肿) 癌症患者)已被认为是LAS的危险因素，对其潜在机制知之甚少 调节LM向LAS的进程。拟议研究的长期目标是了解 LM向LAS进展的分子和细胞机制以开发有效的新策略 对这种致命疾病的预防和治疗。在之前的资助期，我们开发了一个老鼠模型， 可诱导EC特异性缺失Tsc1肿瘤抑制基因(Tsc1iΔEC小鼠)，概括显著 人类LAS的特征。使用来自Tsc1iΔEC小鼠的一种新的肿瘤细胞系，我们进一步表明 敲除或敲除不同自噬基因阻断自噬抑制血管肿瘤细胞 体外增殖和体内致瘤性。转录特征分析和额外的机制分析 提示骨桥蛋白的自噬依赖性表达及其潜在的自分泌刺激的作用 JAK/STAT3信号在促进肿瘤细胞增殖和致瘤性中的作用更重要的是，通过生成 在Tsc1 iΔEC模型中EC特异的自噬基因FIP200的双重条件敲除，我们发现 自噬抑制，虽然不影响最初的LM发育，但阻止了LM向LAS的进展。我们也 制备了一种新的TSC1/FIP200-4A双条件敲击小鼠，为进一步研究其作用机制奠定了基础 FIP200介导的体内自噬对LM向LAS进展的调节此外，我们还进行了基因组测试- CRISPR-Cas9广谱筛选与血管肿瘤相关基因的筛选 体内生长，包括编码RAS信号负调控因子的RASA1，暗示潜在的协同作用 RAS/MAPK和mTORC1信号在LM的发育和向LAS的进展之间。基于这些强大的 支持数据，并使用我们独特的鼠标和细胞模型，我们建议1)确定 自噬对血管肿瘤细胞增殖和致瘤性的调节；2)检测自噬的靶向性 以及它的下游途径，以阻止LM进展到LAS；以及3)探索 RAS和mTORC1信号在血管肿瘤发生发展中的作用总而言之，这些研究 将极大地促进我们对LM向LAS进展的分子和细胞机制的理解， 这可能有助于未来为这种毁灭性疾病设计有效的预防和新的治疗方法。

Summary/Abstract Endothelial cells (ECs) are key components of both blood and lymphatic vessels, and aberrant EC proliferation due to abnormalities in various signaling pathways contributes to a wide range of vascular anomalies including vascular malformation and tumors. Lymphatic malformation (LM) and lymphangiosarcoma (LAS) are vascular anomalies originating from lymphatic ECs. While LM mostly remains as a benign disease, a fraction of LM patients progress to the highly aggressive and deadly LAS. Although LM (e.g. chronic lymphedema in breast cancer patients) has been recognized as a risk factor for LAS, very little is known about underlying mechanisms regulating the progression of LM to LAS. The long-term goal of the proposed studies is to understand the molecular and cellular mechanisms of LM progression to LAS in order to develop new strategies for effective prevention and treatment of this deadly disease. In prior funding periods, we developed a mouse model with inducible EC-specific deletion of Tsc1 tumor suppressor gene (Tsc1iΔEC mice), which recapitulates salient features of human LAS. Using a new tumor cell line derived from Tsc1iΔEC mice, we further showed that autophagy blockade by knockout or knockdown of different autophagy genes inhibited vascular tumor cell proliferation in vitro and tumorigenicity in vivo. Transcriptional profiling and additional mechanistic analysis suggested a role for the autophagy-dependent expression of osteopontin and its potential autocrine stimulation of Jak/Stat3 signaling in promoting tumor cell proliferation and tumorigenicity. More importantly, by generating EC-specific double conditional knockout of an essential autophagy gene Fip200 in Tsc1iΔEC model, we found that autophagy inhibition, while not affecting the initial LM development, blocked LM progression to LAS. We also prepared a new Tsc1/Fip200-4A double conditional knockin mouse for further analysis of the mechanisms of regulation of LM progression to LAS by FIP200-mediated autophagy in vivo. In addition, we performed a genome- wide CRISPR-Cas9 screen and identified several genes with tumor suppressive functions for vascular tumor growth in vivo, including Rasa1 encoding a negative regulator of Ras signaling, suggesting potential synergy between Ras/MAPK and mTORC1 signaling in LM development and progression to LAS. Based on these strong supporting data and using our unique mouse and cell models, we propose to 1) determine the mechanisms of autophagy regulation of vascular tumor cell proliferation and tumorigenicity; 2) examine targeting of autophagy and its downstream pathways to block LM progression to LAS; and 3) explore the potential synergy between Ras and mTORC1 signaling in the development and progression of vascular tumors. Together, these studies will significantly advance our understanding of the molecular and cellular mechanisms of LM progression to LAS, that may contribute to the future design of effective prevention and novel therapies for this devastating disease.