第一部分摘要：

背景：嘧啶核苷酸从头合成的增强是肿瘤代谢重编程的标志之一，其中二氢乳清酸脱氢酶（Dihydroorotate Dehydrogenase，DHODH）是嘧啶从头合成（pyrimidine de novo synthesis）中的关键分子，因此成为治疗肿瘤的新兴靶点。然而前期DHODH抑制剂在结直肠癌（colorectal cancer，CRC）及肝癌（hepatocarcinoma，HCC）的II期临床试验疗效微弱，因此探究肿瘤细胞对DHODH抑制剂耐药性的机制十分重要。尽管有研究表明核苷酸转运体和自噬通路的活化可能参与对DHODH抑制剂的耐药性，但药物组合的疗效并不显著，提示存在其它的耐药性机制。巨胞饮是一种非选择性的、依赖肌动蛋白的内吞作用，能在营养匮乏的微环境中提供营养物质维持肿瘤的生长。因此，本研究旨在探究巨胞饮在肿瘤细胞对DHODH抑制剂耐药性的作用及机制，为结直肠癌和肝癌患者提供新的治疗策略。

方法：利用GSEA分析、鬼笔环肽染色、透射电镜检测、DQ-BSA染色、qRT-PCR和70kDa葡聚糖摄取实验探究抑制DHODH后肿瘤细胞巨胞饮的变化。利用克隆形成实验、细胞计数实验和3D成球实验探究DHODH抑制剂与巨胞饮抑制剂或EGFR抑制剂联合使用对肿瘤细胞增殖的影响。利用结直肠癌细胞HT29和肝癌细胞Huh7皮下移植瘤裸鼠模型以及AOM/DSS诱导的结直肠癌模型研究DHODH抑制剂联合巨胞饮或EGFR抑制剂的协同抗肿瘤作用。通过生物信息学分析和肿瘤患者组织微阵列芯片的免疫组化染色验证临床样本中DHODH-p65-EREG/EGFR-PAK1信号轴。

结果：DHODH抑制剂诱导的内源性核苷酸缺乏激活巨胞饮。肿瘤细胞通过巨胞饮补充核苷酸维持在DHODH抑制剂存在时细胞的生长，而核苷酸转运体和自噬不参与DHODH抑制剂的耐药性。进一步机制探究表明：DHODH抑制剂通过NF-κB转录EREG促进EGFR-PAK1的活化。体内体外联合DHODH抑制剂及EGFR抑制剂协同抑制结直肠癌和肝癌生长。此外在结直肠癌和肝癌患者中，DHODH的表达水平与NF-κB-EGFR-PAK1信号轴呈显著负相关。

结论：DHODH抑制剂引起的内源性核苷酸缺乏可诱导肿瘤细胞通过NF-κB-EGFR-PAK1信号轴介导的巨胞饮提供核苷酸维持其生长。联合DHODH抑制剂与巨胞饮抑制剂或EGFR抑制剂，能在体内及体外显著抑制肿瘤生长。

第二部分摘要：

背景：非小细胞肺癌（non-small cell lung cancer，NSCLC）是肺癌的主要类型，其中肺腺癌（lung adenocarcinoma，LUAD）是非小细胞肺癌最常见的亚型。由于LUAD往往在晚期才被诊断出来，导致其发病率和死亡率持续上升。因此深入了解LUAD的发病机制对于其诊断和治疗十分重要。表观遗传改变是癌症发生的重要因素之一。N6-甲基腺苷（m6A）RNA的活性和可逆性改变对控制基因活性和决定细胞命运至关重要。然而，甲基化转移酶KIAA1429（VIRMA）在LUAD中的表达水平及功能仍不明确。

方法：使用克隆形成及Transwell等实验明确KIAA1429在LUAD中的功能。使用转录组测序、RNA甲基化免疫共沉淀高通量测序（MeRIP-seq）以及RNA稳定性检测等方法明确及检测下游靶基因的半衰期和稳定性。

结果：KIAA1429在肺腺癌中高表达并且显著促进LUAD细胞的增殖和转移。转录组测序及MeRIP-seq分析表明，KIAA1429可促进下游靶基因ARHGAP30 mRNA中m6A的水平，从而降低其稳定性和表达。更重要的是，KIAA1429通过下调ARHGAP30的表达从而活化PI3K/AKT信号通路，进而促进肺腺癌细胞的增殖和转移。

结论：KIAA1429促进ARHGAP30 mRNA的m6A的水平进而下调其表达，从而促进PI3K/AKT通路介导的肺腺癌细胞的增殖和转移。

Part ⅠAbstract：

Background: The upregulation of pyrimidine nucleotide de novo synthesis is a distinguishing feature of tumor metabolic reprogramming. Among these alterations, dihydroorotate dehydrogenase (DHODH) plays a pivotal role in pyrimidine de novo synthesis, making it a promising target for tumor treatment. Nevertheless, the effectiveness of DHODH inhibitors in phase II clinical trials for colorectal cancer (CRC) and hepatocellular carcinoma (HCC) was limited during the initial phase. Hence, it is essential to investigate the mechanism of tumor cell resistance to DHODH inhibitors. While research has indicated that the activation of nucleotide transporters and autophagy pathways could play a role in resistance to DHODH inhibitors, the effectiveness of drug combinations appears to be limited, indicating the presence of additional resistance mechanisms. Macropinocytosis is a form of non-selective, actin-dependent endocytosis that facilitates the uptake of nutrients to support tumor growth in microenvironments with limited nutrient availability. Hence, the primary objective of this study is to investigate the function and mechanism of macropinocytosis in conferring tumor cell resistance to DHODH inhibitors. These results also aim to propose novel treatment approaches for patients with colorectal cancer and hepatocellular carcinoma.

Methods: The investigation into the alterations in tumor cell macropinocytosis following the inhibition of DHODH involved various methods including GSEA analysis, phalloidin staining, transmission electron microscopy, DQ-BSA staining, qRT-PCR and 70 kDa TMR-dextran uptake assay. Cloning forming assays, cell counting assays and 3D spheroidization assays were conducted to evaluate the effects of combined application of DHODH inhibitors with EGFR or macropinocytosis inhibitors on tumor cell proliferation. Utilizing HT29 and Huh7 subcutaneous tumor models in nude mice, as well as AOM/DSS models, this research investigates the in vivo synergistic anti-tumor effects of DHODH inhibitors in combination with EGFR or macropinocytosis inhibitors. The validation of the DHODH-p65-EREG/EGFR-PAK1 signaling axis in clinical samples involved bioinformatics analysis and immunohistochemical staining of tumor patient tissue microarray chips.

Results: The inhibition of DHODH results in endogenous nucleotide deficiency, which triggers the activation of macropinocytosis. Tumor cell proliferation in the presence of DHODH inhibitors is maintained through macropinocytosis, which supplies extra nucleotides. Nucleotide transporters and autophagy are not involved in mediating resistance to DHODH inhibitors. Further exploration of the mechanism elucidates that DHODH inhibitors exert their effects by modulating NF-κB transcription of EREG, which in turn enhances the activation of EGFR-PAK1. The concurrent use of DHODH inhibitors and EGFR inhibitors has been shown to synergistically impede the proliferation of colorectal and liver cancer cells, as evidenced in both in vitro and in vivo studies. A notable inverse correlation was identified between DHODH and the NF-κB-EGFR-PAK1 signaling axis in patients with colorectal and liver cancer.

Conclusion: The depletion of endogenous nucleotides resulting from DHODH inhibitors can trigger tumor cells to activate the NF-κB-EGFR-PAK1 signaling axis, facilitating macropinocytosis to acquire nucleotides essential for sustaining their proliferation. The concurrent administration of DHODH inhibitors with either macropinocytosis inhibitors or EGFR inhibitors has been shown to markedly impede tumor proliferation both in vivo and in vitro.

Part Ⅱ Abstract：

Background: Non-small cell lung cancer (NSCLC) is the main type of lung cancer, of which lung adenocarcinoma (LUAD) is the most common subtype of NSCLC. Because LUAD is often diagnosed at an advanced stage, its morbidity and mortality continue to increase. Therefore, a thorough understanding of the pathogenesis of LUAD is important for its diagnosis and treatment. Epigenetic alterations are one of the important factors in cancer development, and changes in the activity and reversibility of N6-methyladenosine (m6A) RNA are critical for controlling gene activity and determining cell fate. However, the expression level and function of the methylation transferase KIAA1429 (VIRMA) in LUAD remain unclear.

Methods: The function of KIAA1429 in LUAD was elucidated through the utilization of experimental techniques, including clone formation and Transwell assays. The half-life and stability of downstream target genes were elucidated and validated through transcriptome sequencing, methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA stability assay.

Results: KIAA1429 exhibits high expression levels in lung adenocarcinoma and plays a significant role in promoting the proliferation and metastasis of LUAD cells. Transcriptome sequencing and MeRIP-seq analysis revealed that KIAA1429 has the capability to elevate the m6A level in ARHGAP30 mRNA, consequently reducing its stability and expression. More significantly, KIAA1429 facilitates the proliferation and metastasis of lung adenocarcinoma cells by suppressing ARHGAP30, thereby activating the PI3K/AKT signaling pathway.

Conclusion: KIAA1429 facilitates the m6A modification of ARHGAP30 mRNA, resulting in reduced expression, which enhances the proliferation and metastasis of lung adenocarcinoma cells by activating the PI3K/AKT pathway.