Functional contributions of glycogen metabolism to ovarian cancer metastasis

项目概要/摘要 高级别浆液性卵巢癌 (OvCa) 的生物学特性与大多数上皮性肿瘤不同， 因为血行转移很少见。卵巢肿瘤仍局限于腹膜腔 主要播种到网膜和腹膜。在那里，癌细胞与癌症相关成纤维细胞相互作用 （CAF）促进癌细胞增殖、侵袭和转移。研究双向信号 在这两种细胞类型之间，我们将它们共培养并使用 SILAC 结合定量、无标记 磷酸蛋白质组学可识别 OvCa 细胞和成纤维细胞中的磷酸酪氨酸信号转导事件。我们 确定了癌细胞中磷酸葡萄糖变位酶 1 (PGM1) 的激活，指出了调节 CAF 在 OvCa 细胞中进行糖原代谢。此外，初步实验表明，糖原分解 为癌细胞提供能量并调节蛋白质糖基化和组蛋白甲基化。基于这些 数据显示，该应用的主要假设是 CAF 介导的糖原分解促进 通过几种不同的机制进行转移，包括糖酵解、蛋白质糖基化和表观遗传学。 在目标 I 中，我们将探讨 OvCa 细胞和 PGM1 激活或抑制的代谢后果。 使用非靶向代谢组学和代谢流研究对正常输卵管上皮细胞进行研究。我们将使用 PGM 敲低细胞并使用 PGM1 抑制对粘附/侵袭/增殖的影响 3D 模型和同基因小鼠转移模型。然后我们将利用高通量 RNAi 文库 目标是确定响应 CAF 调节糖原分解的上游信号通路。瞄准 II、我们将利用人类蛋白质糖基化系统地研究糖原介导的糖基化事件 阵列来识别哪些蛋白质被 CAF 介导的糖原分解特异性糖基化。这个 确定糖基转移酶如何调节 CAF-的研究将补充调查 介导的糖基化。然后，我们将使用以下方法研究糖基转移酶在体内转移测定中的作用 原代 CAF/OvCa 细胞和 OvCa 转移的同基因小鼠模型。使用靶向代谢组学 对与 OvCa 细胞共培养的 CAF 的数据和通量分析，我们发现糖原分解诱导 α- 酮戊二酸（KG）。因此，我们在目标 III 中建议研究糖原分解如何改变表观遗传变化 癌细胞。我们将表征组蛋白甲基化 (ChIP-seq) 和 DNA 的全基因组变化 羟甲基化 (Nano-hmC-Seal) 来识别受糖原分解表观遗传调节的基因。的 拟议的实验旨在确定糖原分解和表观遗传学之间的联系，以识别功能 OvCa 转移的调节因子。通过了解肿瘤器官中的糖原代谢，我们也许能够 阐明对转移很重要的新代谢机制，这可能导致鉴定 治疗转移性卵巢癌的新的临床相关方法。

PROJECT SUMMARY/ABSTRACT The biology of high grade serous ovarian cancer (OvCa) is distinct from that of most epithelial tumors, in that hematogenous metastases are rare. Ovarian tumors remain confined to the peritoneal cavity and primarily seed to the omentum and peritoneum. There, cancer cells interact with cancer associated fibroblasts (CAFs) which promote cancer cell proliferation, invasion, and metastasis. To study bidirectional signaling between the two cell types, we co-cultured them and used SILAC coupled with quantitative, label-free phosphoproteomics to identify phospho-tyrosine signaling events in both OvCa cells and fibroblasts. We identified activation of phosphoglucomutase 1 (PGM1) in the cancer cells, pointing towards regulation of glycogen metabolism in OvCa cells by CAFs. Further, preliminary experiments showed that glycogenolysis provides energy to cancer cells and regulates protein glycosylation and histone methylation. Based on these data, the primary hypothesis underlying this application is that CAF-mediated glycogenolysis promotes metastasis through several different mechanisms, including glycolysis, protein glycosylation, and epigenetics. In Aim I, we will explore the metabolic consequences of PGM1 activation or inhibition in both OvCa cells and normal fallopian tube epithelial cells using untargeted metabolomics and metabolic flux studies. We will use PGM knockdown cells and investigate the effects of PGM1 inhibition on adhesion/invasion/proliferation using a 3D model and a syngeneic mouse model of metastasis. We will then utilize a high-throughput RNAi library with the goal of identifying the upstream signaling pathways regulating glycogenolysis in response to CAFs. In Aim II, we will systematically study glycogen-mediated glycosylation events using human protein glycosylation arrays to identify which proteins are specifically glycosylated by CAF-mediated glycogenolysis. This investigation will be complemented by studies determining how glycosyltransferase enzymes regulate CAF- mediated glycosylation. We will then study the role of glycosyltransferases on in vivo metastasis assays using primary CAF/OvCa cells and a syngeneic mouse model of OvCa metastasis. Using targeted metabolomics data and flux analysis of CAFs co-cultured with OvCa cells, we discovered that glycogenolysis induced α- ketoglutarate (KG). We therefore propose, in Aim III, to study how glycogenolysis alters epigenetic changes in cancer cells. We will characterize genome-wide changes in histone methylation (ChIP-seq) and DNA hydroxymethylation (Nano-hmC-Seal) to identify genes epigenetically regulated by glycogenolysis. The proposed experiments aim to define the link between glycogenolysis and epigenetics to identify functional regulators of OvCa metastasis. By understanding glycogen metabolism in the tumor organ, we may be able to elucidate novel metabolic mechanisms important for metastasis, which could result in the identification of a new and clinically relevant approach to the treatment of metastatic ovarian cancer.