Analysis of Pten-null primary mouse astrocytes revealed common differentially expressed genes with human glioblastoma transcriptomes

Ph.D.Glioblastoma, also known as Glioblastoma multiforme (GBM), is the most lethal and frequent brain tumor. Despite the most advanced therapy, GBM recurs frequently. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene whose alteration is one of the most frequent events in human cancers. PTEN possesses a lipid phosphatase activity and negatively regulates the phosphoinositide 3-kinase (PI3K) pathway. Cumulative evidence suggests that PTEN alterations play pivotal roles in tumorigenesis of GBM as 60% of GBM patients acquired PTEN mutations, and 80% of GBM patients had PTEN protein loss.Transcriptomic profiling of primary astrocytes derived from Pten knockout (KO) mice revealed an overlapping enrichment of mitotic regulators and cell-cycle related pathways with human GBM. A total of 60 overlapping DEGs from enriched pathways were selected for further RT-qPCR validation. 4 genes (RCC1, RCC2, NEK2 and CHK2) were significantly upregulated in Pten KO astrocytes. Upregulated RCC1 and RCC2 expression were tightly associated with brain cancer histological grades and prognosis. Therefore, PTEN may play a role in gliomagenesis via regulating RCC1 and RCC2.Previous studies reported that RCC1 and RCC2 play roles in tumorigenesis. Coincidently, our study showed that silencing RCC1 or RCC2 could suppress GBM cell proliferation. Both RCC1 and RCC2 belong to the RCC1 superfamily, which contains the RCC1-like domain (RLD). Regulator of chromatin condensation 1 (RCC1) has been reported as a major guanine-nucleotide exchange factor (GEF) for Ran GTPase and participates in various biological processes such as mitosis. RNA-seq analysis identified 736 differentially expressed genes (DEGs) between control siRNA- and RCC1 siRNA-treated A1172 cells. Gene Set Enrichment Analysis (GSEA) analysis suggested that RCC1 silencing upregulates the apoptosis pathway.Regulator of chromatin condensation 2 (RCC2) has been reported to be a GEF for Rac1 or RalA GTPase and regulates cell cycle progression. Using RNA-seq, we identified 737 DEGs between control siRNA- and RCC2 siRNA-treated A1172 cells. GSEA analysis delineated G2- M progression and glucose metabolism as the most notably altered pathway by silencing RCC2. Indeed, we observed a G2-M arrest in cells with RCC2 silenced, suggesting that RCC2 is essential for G2-M progression. We also found that RCC2 may regulate glucose metabolism via hexokinase 2 (HK2), which is a key driver of metabolic regulation. Both the RCC2 silencing model and the RCC2 overexpression model demonstrated that RCC2 possesses a positive association with HK2 expression levels. Additionally, RCC2 can affect metabolic pathways. We found that RCC2 knockout cells have more dependency on oxidative phosphorylation and relatively less dependency on glycolysis to get energy.The connectivity map (C-map) analysis was conducted and identified cephaeline as a promising inhibitor for RCC2-associated gene signatures. RCC2 knocking down and cephaeline synergistically inhibit glioblastoma cell viability.In summary, our study suggested that PTEN loss might promote tumorigenesis of glioblastoma via upregulating RCC1 and RCC2. We provided evidence that RCC2 exerts a positive effect on G2-M progression as well as glycolytic metabolism. These findings may shed light on anti-RCC2 targeted therapy in GBM treatment.膠質母細胞瘤，又稱稱多形性膠質母細胞瘤（縮寫：GBM），是最常見且惡性度最高的腦腫瘤。儘管有最先進的治療方法，但膠質母細胞瘤仍經常復發。磷酸酯酶與張力蛋白同源物是（PTEN）是壹種抑癌基因，其改變在癌癥中尤為常見。 PTEN具有脂質磷酸酶活性，並負調節磷酸肌醇3激酶（PI3K）途徑。證據表明，PTEN的改變在膠質母細胞瘤（GBM）的腫瘤發生中起關鍵作用， 60％的GBM 患者中有PTEN 突變，而80％的GBM患者俱有PTEN蛋白丟失。Pten基因敲除小鼠的原代星形膠質細胞的轉錄組譜與人類GBM轉錄組譜比對後我們發現，比對重合基因富集於有絲分裂相關的通路。我們從富集的有絲分裂相關的通路中選取了60個基因進行進壹步的RT-qPCR驗證。其中有4個基因（RCC1，RCC1，NEK2和CHK2）在Pten敲除組中顯著上調。 RCC1和RCC2與腦癌的組織學分級和預後密切相關。因此，PTEN可能通過調節RCC1和RCC2在神經膠質瘤發生中起作用。先前的研究報導RCC1 和RCC2 參與了腫瘤發生。同時，我們的研究表明沈默RCC1或RCC2可以抑制GBM細胞增殖。 RCC1和RCC2都屬於RCC1超家族，它們都包含RCC1樣域（RLD）。RCC1已被報導為Ran GTPase的鳥嘌呤-核苷酸交換因子（GEF），並參與各種生物過程，例如有絲分裂。通過RNA-seq分析對對照組和RCC1沈默組的A1172細胞進行比對並得到了736個差異表達基因。GSEA分析表明，RCC1沈默可上調細胞雕亡相關途徑。據報導， RCC2是Rac1或RalA GTPase的鳥嘌呤-核苷酸交換因子，可調節細胞週期進程。通過RNA-seq分析對對照組和RCC2沈默組的A1172細胞進行比對，得到了737個差異表達基因。GSEA分析表明，沈默RCC2改變了G2-M進程和糖代謝。實際上，我們在RCC2沈默細胞中觀察到G2-M停滯，表明RCC2是G2-M進展必需條件。我們還發現RCC2可能通過己糖激酶2（HK2）調節葡萄糖代謝，而己糖激酶2是代謝調節的關鍵驅動力。 RCC2沈默模型和RCC2過表達模型均證明RCC2與HK2呈正相關。此外，RCC2可以影響代謝途徑。我們發現RCC2基因敲除細胞對氧化磷酸化的依賴性更大，而對從糖酵解獲得能量的依賴性相對較小。通過C-map 分析，我們發現頭孢菌素是RCC2 相關基因的潛在的抑製劑。 RCC2沈默和頭孢菌素以協同方式抑製膠質母細胞瘤細胞活力。總之，我們的研究證明PTEN缺失可能通過上調RCC1和RCC2促進膠質母細胞瘤的發生。我們提供的證據表明，RCC2影響了G2-M進程以及糖酵解代謝。這些發現可能為GBM治療中的抗RCC2靶向治療提供了啟示。Liu, Tian.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 147-162).Abstracts also in Chinese.Title from PDF title page (viewed on February 11, 2022)