第一部分 苹果酸酶1在乳腺癌中的临床意义及其功能研究 背景：苹果酸酶1(Malic enzyme 1，ME1)是一种催化乳酸生成丙酮酸的酶，可以促进糖酵解环节的进行，在肿瘤的Warburg效应中发挥重要的作用。尽管有研究发现ME1可以促进某些肿瘤的进展，它在乳腺癌中的功能尚不清楚。 方法：我们利用包含220例病人样本的组织芯片，通过免疫组化技术评估ME1在这些样本中的表达水平，并且分析ME1表达水平和一些临床病理特征的相关性。进一步采用生存分析的方法探索ME1的预后作用。此外，我们通过蛋白免疫印迹法和免疫荧光技术评估ME1在乳腺癌细胞中的定位和表达情况，通过细胞增殖检测、平板克隆形成、transwell迁移和侵袭实验探索ME1在乳腺癌细胞中的功能，并通过检测活性氧(Reactive oxygen species，ROS)水平初步探索ME1发挥作用的机制。 结果：在乳腺癌组织中，ME1高表达与以下特征具有相关性：肿瘤>2㎝(P=0.036)、淋巴结转移阳性(P<0.001)、脉管浸润阳性(P=0.001)。生存分析提示ME1高表达的患者无复发生存期(Recurrence free survival，RFS)更短(P=0.010)。多因素分析进一步发现ME1表达水平是RFS的独立预后因素(P=0.029，HR=5.343[1.19， 23.97])。与Luminal型乳腺癌细胞相较，人表皮生长因子-2阳性的乳腺癌细胞和三阴性乳腺癌细胞高表达ME1。在MCF-7细胞中上调ME1可以显著增强细胞的增殖能力和转移能力、促进上皮细胞-间充质转化(Fpithelial-mesenchymal transition，MET)发生、并且引起ROS水平的下降；在MAD-MB-468细胞中下调ME1可以显著降低细胞的增殖能力和转移能力、抑制MET发生、并且引起ROS水平的上升。用过氧化氢对过表达ME1的MCF-7细胞进行预处理，可以上调细胞的ROS水平，并且逆转过表达ME1的促转移功能；用N-乙酰半胱氨酸对敲低ME1的MAD-MB-468细胞进行预处理，可以下调细胞的ROS水平，并且逆转敲低ME1的抑制转移效应。 结论：ME1在乳腺癌中高表达提示患者预后不良；ME1可以增强乳腺癌细胞的增殖能力和转移能力、介导ME1发生；ME1通过降低ROS水平发挥部分促癌作用。 关键词：苹果酸酶1；乳腺癌；预后；癌基因；活性氧 中图分类号：R737.9 第二部分 免疫微环境分型在乳腺癌中的临床意义及预后价值 背景：免疫检查点抑制剂近来在晚期乳腺癌中展示出了良好的治疗效果，但是，仅有少部分患者能够从中获益。为了更加系统地认识肿瘤免疫、为免疫治疗选择潜在的获益人群，我们利用免疫组学数据描绘了乳腺癌的免疫微环境。 方法：我们分析了TCGA中1092例乳腺癌患者的表达谱数据。利用ESTIMATE中的141个免疫相关基因，采用Kmeans聚类法将患者分为2类——免疫浸润型和非免疫浸润型。进一步利用CIBERSORT和MCP-Counter阵列中代表微环境细胞类型和免疫状态的基因，采用Kmeans聚类法将免疫浸润型患者再分为2类——免疫激活型和免疫耗竭型。我们评估了免疫微环境分型与患者临床病理特征和预后的相关性，并利用GEO数据库验证了免疫微环境分型的预后价值。 结果：在TCGA队列中，60.3%(659/1092)的患者被划分为非免疫浸润型，39.7% (433/1092)的患者被划分为免疫浸润型。进一步对免疫浸润型患者进行分类，结果显示16.8%。(184/1092)的患者为免疫激活型，22.8%(249/1092)的患者为免疫耗竭型。淋巴结没有转移、肿瘤分期早和激素受体阴性的患者倾向于免疫激活型(P<0.05)。在单因素生存分析中，免疫激活型提示患者有更好的无病生存期(Disease free survival， DFS)(P=0.019)和总体生存期(Overall survival，OS)(P=0.041)。在多因素COX模型中校正肿瘤大小、淋巴结转移情况、激素受体状态和HER-2状态，显示免疫激活型是患者DFS(P=0.006，HR=0.177[0.051,0.610])和OS(P=0.032，HR=0.370 [0.149，0.919])的独立预后因素。在GEO队列中，免疫激活型提示患者有更好的RFS (P=0.029)和OS(P=0.026)，在新辅助化疗中有更高的病理完全缓解率(P=0.001)。此外，免疫激活型患者有较高的ME1表达水平(P=0.047)；并且，在非免疫激活型患者中，ME1高表达提示较差的近期预后(P=0.047)。 结论：我们将乳腺癌的免疫微环境分为3种类型——免疫激活型、免疫耗竭型和非免疫浸润型。免疫激活型患者有较好的DFS和OS，在新辅助化疗中有更高的病理完全缓解率。免疫微环境分型可以提示肿瘤发展潜在的免疫学机制，对乳腺癌患者的免疫治疗有一定的指导意义。 关键词：免疫微环境；乳腺癌；预后 中图分类号：R737.9

 Part 1 Clinical Significance and Biological Function of Malic Enzyme 1 in Breast Cancer Background: Malic enzyme 1 (ME1) catalyzes malate to pyruvate and thus promotes glycolysis, playing an important role in the Warburg effect. Although studies have suggested its potential role in tumor progression, its function in breast cancer remains unclarified. Methods: We evaluated ME1 expression in 220 patients with tissue microarray-based immunohistochemistry and explored the relationships between ME1 expression and clinicopathological features. Survival analyses were further performed to determined its prognostic value. Moreover, we profiled ME1 location and expression in breast cancer cell lines via western blot and Immunofluorescence, and then focused on its role in cell viability and motility via Cell counting kit-8 (CCK-8), colony formation, transwell migration and invasion assays. Reactive oxygen species (ROS) was detected by dihydroethidium (DHE) and 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) to tentatively investigate the underlying mechanism. Results: In breast cancer tissues, high ME1 expression was significantly associated with larger tumor size (P=0.036),lymph node metastasis (P<0.001) and lymph-vascular invasion (P=0.001). Survival analysis showed high ME1 expression was significantly correlated with worse recurrence free survival (RFS) (P=0.010). Multivariate analysis further identified high ME1 expression as an independent prognostic factor for RFS (P= 0.029,HR=5.343 [1.19,23.97]). In vitro, human epidermal growth factor receptor-2 positive and triple negative breast cancer cell lines showed high expression of ME1, while Luminal cell lines showed low expression of ME1. Upregulation of ME1 remarkably enhanced viability and motility, induced epithelial-mesenchymal transition (EMT), and decreased ROS level in MCF-7 cells. Its knockdown had a profound inhibitory effect on viability, motility and EMT, and increased ROS level in MDA-MB-468 cells. When pretreated with hydrogen peroxide, an oxidizing agent, MCF-7 cells overexpressing ME1 lost its motility. Meanwhile, MDA-MB-468 cells with knock-down of ME1 restored its motility when pretreated with N-acetyl cysteine, an antioxidant. Conclusion: Our work first indicated that ME1 was a promising negative prognostic biomarker in breast cancer. In vitro, ME1 could enhance viability and motility of breast cancer cells partially via decreasing ROS level. Keywords: Malic enzyme 1; Breast cancer; Prognosis; Oncogene; Reactive oxygen species Chinese Library Classification: R737.9 Part 2 Clinical Significance and Prognostic Value of Immune Microenvironment Subtypes in Breast Cancer Background: Checkpoint blockade immunotherapy has been reported to show promising responses in metastatic breast cancer. However, only a small quantity of patients benefited from immunotherapy. To understand the underlying mechanisms of immunotherapy and distinguish appropriate patients, we profiled the immune microenvironment landscape of breast cancer. Methods: We analyzed 1092 breast cancer samples from The Cancer Genome Atlas (TCGA) profiled by RNA sequencing. Using 141 immune-related genes from ESTIMATE, the cohort was divided into two subtypes, Immune Subtype and non-Immune Subtype, by Kmeans cluster analysis. Immune-related gene signatures from CIBERSORT and MCP-Counter representing different cell types and immune status were used to profile the Immune Subtype by Kmeans cluster analysis, and we further classified them into Active Immune Subtype and Exhausted Immune Subtype. We explored the associations between clinicopathological parameters and immune microenvironment subtypes. Prognostic value of immune microenvironment subtypes was evaluated in TCGA, and further validated in a cohort consisting of 1861 patients from GEO datasets. Results: We identified 60.3% (659/1092) of breast cancer in TCGA dataset as non-Immune Subtype and 39.7% (433/1092) as Immune Subtype. Further, we classified the Immune Subtype into two distinct subtypes, Active and Exhausted Immune Subtypes (16.8% and 22.8%, respectively). Compared with the other two subtypes, Active Immune Subtype was associated with no lymph node metastasis, early tumor stage and negative hormone receptor (all P <0.05). In survival analyses, Active Immune Subtype was significantly correlated with favorable disease-free survival (DFS) (P=0.019) and overall survival (OS) (P=0.041). Adjusting for tumor size, lymph node, hormone receptor and human epidermal growth factor receptor-2 status, multivariate analyses further identified Active Immune Subtype as an independent prognostic factor for DFS (P=0.006,HR=0.177 [0.051, 0.610]) and OS (P=0.032, HR=0.370 [0.149, 0.919]). In GEO cohort, Active Immune Subtype also correlated with favorable relapse-free survival (P=0.029) and OS (P=0.026). In addition, Active Immune Subtype predicted higher pathologic complete response rate in neoadjuvant chemotherapy (P=0.001). Furthermore, Active Immune Subtype correlated with higher ME1 expression level (P<0.019).For non-Active Immune Subtype patients, high ME1 expression indicated worse short-term prognosis (P=0.047). Conclusion: Our work classified breast cancer into three immune microenvironment subtypes, including Active Immune Subtype, Exhausted Immune Subtype and non-Immune Subtype. Active Immune Subtype indicated favorable DFS and OS, and predicted higher pathologic complete response rate in neoadjuvant chemotherapy. This novel classification can reveal the underlying immunological mechanisms of tumor progression and may assist immunotherapy decision-making for breast cancer patients. Keywords: Immune microenvironment subtype; Breast cancer; Prognosis Chinese Library Classification: R737.9