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）的生物学不同于大多数上皮性肿瘤， 因为血行转移是罕见的。卵巢肿瘤仍然局限于腹膜腔， 主要植入网膜和腹膜在那里，癌细胞与癌症相关的成纤维细胞相互作用， 在某些实施方案中，癌细胞因子（CAF）促进癌细胞增殖、侵袭和转移。研究双向信号 在两种细胞类型之间，我们将它们共培养，并使用SILAC与定量，无标记 磷酸蛋白质组学以鉴定OvCa细胞和成纤维细胞中的磷酸酪氨酸信号传导事件。我们 鉴定了癌细胞中磷酸葡萄糖变位酶1（PGM 1）的激活，指出调节 通过CAF在OvCa细胞中的糖原代谢。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 根据数据，本申请的主要假设是，CAF介导的糖原分解促进 转移通过几种不同的机制，包括糖酵解，蛋白糖基化和表观遗传学。 在目的I中，我们将探索PGM 1激活或抑制在OvCa细胞中的代谢后果， 正常输卵管上皮细胞使用非靶向代谢组学和代谢通量研究。我们将使用 PGM基因敲减细胞，并研究PGM 1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， 目的是确定上游信号通路调节糖原分解反应CAFs。在Aim中 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，以确定哪些蛋白质是由CAF介导的糖原分解特异性糖基化。这 研究将通过确定糖基转移酶如何调节CAF的研究来补充。 介导的糖基化。然后，我们将研究糖基转移酶在体内转移试验中的作用， 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 数据和与OvCa细胞共培养的CAFs的通量分析，我们发现糖原分解诱导α- 酮戊二酸（KG）。因此，我们建议，在目标III中，研究糖原分解如何改变表观遗传变化， 癌细胞我们将描述组蛋白甲基化（ChIP-seq）和DNA的全基因组变化， 通过使用羟甲基化（Nano-hmC-Seal）来鉴定由糖原分解表观遗传调节的基因。的 提出的实验旨在确定糖原分解和表观遗传学之间的联系，以确定功能性的 OvCa转移的调节剂。通过了解肿瘤器官中的糖原代谢，我们可能能够 阐明新的代谢机制，重要的转移，这可能会导致确定一个 转移性卵巢癌治疗的新的和临床相关的方法。

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.