Novel Markers for Disease Outcome in Breast Cancer

我们继续全面检查非裔美国人和欧裔美国患者乳腺肿瘤的代谢组、蛋白质组和转录组，以发现生物标志物。这种方法的前景是发现预后的标记，以及可能导致非裔美国妇女乳腺癌侵袭的机制。我们描述了67例乳腺肿瘤和邻近非癌组织的代谢组学特征，并描述了200多种代谢物的差异丰度。其中之一是2-羟戊二酸，在雌激素受体主要为阴性的肿瘤中，其含量升高了100倍。2-羟戊二酸的积累与肿瘤中c-Myc信号信号的共同出现密切相关。在后续研究中，我们可以证明乳腺肿瘤主要积聚d -2-羟基戊二酸，并将d -2-羟基戊二酸产生醇脱氢酶含铁蛋白1 （ADHFE1）描述为与疾病生存相关的乳腺癌癌蛋白。我们的数据表明，ADHFE1促进还原性谷氨酰胺代谢，增加d -2-羟戊二酸和线粒体活性氧（ROS）的形成。这导致细胞去分化和上皮间质转化（EMT）增强，从而在高d -2-羟基戊二酸的idh突变癌细胞中发生表型改变。我们还发现d -2-羟戊二酸本身诱导EMT并改变乳腺上皮细胞的代谢，包括谷氨酰胺衍生的α -酮戊二酸的还原羧化和线粒体ROS的上调。将过表达ADHFE1、MYC或ADHFE1和MYC的MCF7人乳腺癌细胞注入小鼠乳腺，证实了ADHFE1作为候选癌基因的功能。ADHFE1和c-Myc的表达不仅以协同方式促进肿瘤生长，而且还增加了肿瘤内4-羟基丁酸盐（4HB）的水平。4HB是ADHFE1产生d -2-羟基戊二酸的底物。该研究的主要发现已于2018年发表（PMID: 29202474）。需要进一步的研究来阐明4HB合成在乳腺癌生物学中的潜在致癌功能。在这项代谢组学研究的延伸中，我们分析了乳腺肿瘤中胆汁酸的存在以及它们的积累与乳腺癌生物学的关系。这一探索性分析揭示了乳腺肿瘤的一个子集积聚胆汁酸，并显示出独特的肿瘤生物学。胆汁酸含量高的肿瘤显示增殖率降低并与生存率提高相关（PMID: 31296531）。这一发现与结肠癌和肝癌的研究结果形成对比，在这两种癌症中，胆汁酸（尤其是去氧胆酸）暴露增加会增加癌症风险，并恶化疾病生物学。在另一项专注于乳腺癌蛋白质组的研究中，我们对乳腺肿瘤进行了综合蛋白质转录组学表征。我们测量了118个人类乳腺肿瘤和邻近非癌组织的整体蛋白质组和转录组表达。比较蛋白质组和转录组数据，我们发现蛋白质组描述了癌组织和非癌组织之间的差异，这些差异没有被转录组捕获。此外，蛋白质组和转录组强调了部分不同的肿瘤生物学。当我们应用这两种技术的综合分析时，该方法揭示了肿瘤中蛋白质- mrna一致性的全球增加。高度相关的蛋白质基因对在蛋白质加工和疾病代谢途径中富集，并且在非裔美国患者的肿瘤中更常见。转录物和蛋白水平之间的一致性增加与侵袭性疾病（包括基底样/三阴性肿瘤）和患者生存率降低进一步相关。我们的研究表明，对癌症中蛋白质组和转录组的综合分析可以揭示超出单一技术能力的疾病特征。这些数据已于2018年发布（PMID: 30501643）。项目2：炎症被广泛认为是癌症进展的诱因。诱导型一氧化氮合酶（NOS2）、环氧化酶-2和胱硫氨酸-合成酶（CBS）是候选炎症标志物，参与伤口愈合、血管生成和癌变。NOS2的上调和一氧化氮（NO）生成的增加也会影响细胞的氧化还原状态，诱导蛋白质、脂质和DNA修饰。我们实验室的研究得出了具有临床意义的新颖观察结果，即NOS2表达与预后基底样转录模式相关，并且是er阴性乳腺肿瘤女性生存不良的独立预测因子。这些发现是与NCI的David Wink博士的实验室合作进行的。这项合作表明，在er阴性乳腺癌细胞中，NOS2的上调发生在缺氧、血清停药、ifn - γ和外源性NO的反应中，这与乳腺癌生物学中肿瘤微环境对NO产生的前馈调节一致。我们目前正在继续这项合作，使用Akoya Codex系统进行多重免疫组化，以确定免疫标记物表达和免疫细胞浸润如何与乳腺肿瘤中NOS2和环氧化酶-2的表达相关。有了这些观察，我们最近开始评估CBS在乳腺癌进展中的作用。这种酶和NOS2一样，释放一种气体信号分子，即硫化氢。硫化氢像NO一样刺激血管生成并可能影响治疗反应。初步资料显示，CBS的产物半胱硫氨酸在乳腺肿瘤中蓄积，在雌激素受体阴性的肿瘤中最高。目前，我们正在建立低、中、高表达CBS的人乳腺癌细胞系，研究CBS对小鼠肿瘤异种移植物生长和转移的剂量效应。我们还将研究CBS对治疗反应和癌症代谢的影响，并使用新型内源性硫化氢荧光探针监测硫化氢信号。项目3：该项目评估环境诱导的应激信号和合并症在乳腺癌进展中的作用。我们开始研究生活压力事件和糖尿病对肿瘤生物学的影响。在一项临床研究中，我们将对计划进行乳腺癌手术的乳腺癌患者进行一项简短的调查，评估他们感受到的压力和社会孤立。我们还将收集这些患者的冷冻肿瘤和邻近正常乳腺组织和血液样本，并评估乳腺组织或血液样本是否具有与他们感知的压力和社会孤立状态相关的生物学特征。我们假设，高感知压力暴露的患者具有与更具侵袭性疾病和较差生存率一致的生物学特征。该试点研究的目的是从同意的患者中收集100对肿瘤/正常配对，并完成调查。在第二项研究中，我们正在评估自我报告的糖尿病与肿瘤生物学和乳腺癌侵袭性之间的关系。在这里，基于调查和医疗记录数据的患者的糖尿病状态将与肿瘤中的整体基因表达和代谢物模式相关联，以确定受糖尿病影响的癌症相关途径。这项研究正在进行中，将评估1型和2型糖尿病是否会诱导肿瘤生物学的变化，从而提高疾病进展的几率。我们特别感兴趣的是代谢途径的变化，以及如何针对它们来减少糖尿病诊断对乳腺癌结果的负面影响。

We continued to comprehensively examine the metabolome, proteome and transcriptome of breast tumors from African-American and European-American patients for biomarker discovery. The promise of this approach is the discovery of markers for prognosis, and of mechanisms that may drive the aggressiveness of breast cancer in African-American women. We characterized the metabolomic profile of breast tumors and adjacent non-cancerous tissue from 67 patients and described the differential abundance of more than 200 metabolites. One of them was 2-hydroxyglutarate, which was elevated up to 100-fold in tumors that were primarily estrogen receptor-negative. This accumulation of 2-hydroxyglutarate was closely associated with the co-occurrence of a c-Myc signaling signature in the tumors. In a follow up study, we could show that breast tumors predominately accumulate D-2-hydroxyglutarate and describe the D-2-hydroxyglutarate-producing alcohol dehydrogenase, iron-containing protein 1 (ADHFE1) as a breast cancer oncoprotein that is associated with disease survival. Our data show that ADHFE1 promotes a reductive glutamine metabolism with increased D-2-hydroxyglutarate and mitochondrial reactive oxygen species (ROS) formation. This leads to cellular de-differentiation and enhanced epithelial-mesenchymal transition (EMT), thereby phenocopying alterations in IDH-mutant cancer cells with high D-2-hydroxyglutarate. We also show that D-2-hydroxyglutarate by itself induces EMT and alters metabolism in mammary epithelial cells, including upregulation of both reductive carboxylation of glutamine-derived alpha-ketoglutarate and mitochondrial ROS. The function of ADHFE1 as a candidate oncogene was corroborated when MCF7 human breast cancer cells overexpressing ADHFE1, MYC, or both ADHFE1 and MYC were injected into the mammary gland of mice. ADHFE1 and c-Myc expression not only enhanced tumor growth in a synergistic manner but also increased intratumor levels of 4-hydroxybutyrate (4HB). 4HB is a substrate for ADHFE1 to produce D-2-hydroxyglutarate. Key findings from this study have been published in 2018 (PMID: 29202474). Additional research is needed to elucidate the potentially oncogenic function of 4HB synthesis in breast cancer biology. In an extension of this metabolome study, we analyzed breast tumors for presence of bile acids and how their accumulation may relate to breast cancer biology. This exploratory analysis revealed that a subset of breast tumors accumulate bile acids and show a distinct tumor biology. Tumors with a high bile acid content showed a decreased proliferation rate and associated with improved survival (PMID: 31296531). This finding contrasts to findings for colon and liver cancer where increased bile acid exposure, especially deoxycholic acid, increases cancer risk and worsens disease biology. In a different study, focusing on the breast cancer proteome, we performed an integrated proteotranscriptomic characterization of breast tumors. We measured global proteome and transcriptome expression in 118 human breast tumors and adjacent non-cancerous tissues. Comparing proteome with transcriptome data, we found that the proteome describes differences between cancerous and non-cancerous tissue that are not captured by the transcriptome. Moreover, the proteome and transcriptome highlighted partially different tumor biologies. When we applied an integrated analysis of both technologies, the approach revealed a global increase in protein-mRNA concordance in tumors. Highly correlated protein-gene pairs were enriched in protein processing and disease metabolic pathways, and occurred more commonly in tumors of African-American patients. The increased concordance between transcript and protein levels was further associated with aggressive disease, including basal-like/triple-negative tumors, and decreased patient survival. Our study indicates that an integrated analysis of the proteome and transcriptome in cancer can uncover disease characteristics beyond the ability of a single technology. These data have been published in 2018 (PMID: 30501643). Project 2: Inflammation is widely recognized as an inducer of cancer progression. Inducible nitric oxide synthase (NOS2), cyclooxygenase-2 and cystathionine-beta-synthase (CBS) are candidate inflammation markers and are involved in wound healing, angiogenesis, and carcinogenesis. NOS2 up-regulation and increased nitric oxide (NO) production also affects the redox state of cells and induces protein, lipid, and DNA modifications. Research by our laboratory led to the novel and clinically significant observation that NOS2 expression is associated with a prognostic basal-like transcription pattern and is an independent predictor of poor survival in women with ER-negative breast tumors. These findings are further pursued in collaboration with the laboratory of Dr. David Wink at the NCI. This collaboration showed that up-regulation of NOS2 in ER-negative breast cancer cells occurs in response to hypoxia, serum withdrawal, IFN-gamma, and exogenous NO, consistent with a feed-forward regulation of NO production by the tumor microenvironment in breast cancer biology. We are currently continuing this collaboration using multiplex IHC with the Akoya Codex system to determine how immune marker expression and immune cell infiltration relates to the expression of NOS2 and cyclooxygenase-2 in breast tumors. Having made these observations, we recently, started to evaluate the role of CBS in breast cancer progression. This enzyme, like NOS2, releases a gaseous signal molecule which is hydrogen sulfide. Hydrogen sulfide like NO stimulates angiogenesis and may affect therapy response. Preliminary data show that cystathionine, a product of CBS, accumulates in breast tumors and are highest in estrogen receptor-negative tumors. Currently, we are establishing human breast cancer cell lines with low, medium and high expression levels of CBS to examine the dose effect of CBS on tumor xenograft growth and metastasis in mice. We will also examine the effects of CBS on therapy response and cancer metabolism and monitor hydrogen sulfide signaling with novel fluorescent probes for endogenous hydrogen sulfide. Project 3: This project evaluates the role of environmentally-induced stress signaling and co-morbidities in breast cancer progression. We started projects studying the impact of stressful life events and diabetes on tumor biology. In a clinical study, we will give breast cancer patients, who are scheduled for breast cancer surgery, a short survey evaluating their perceived stress and social isolation. We will also collect frozen tumor and adjacent normal breast tissue and blood samples from these patients and evaluate whether the breast tissue or the blood samples have a biological signature related to their perceived stress and social isolation status. We hypothesize that patients with a high perceived stress exposure have a biological signature consistent with a more aggressive disease and poorer survival. The pilot study is designed to collect 100 tumor/normal pairs from consented patients with a completed survey. In a second study, we are evaluating the relationship between self-reported diabetes and tumor biology and breast cancer aggressiveness. Here, a patient's diabetes status based on survey and medical record data will be correlated with global gene expression and metabolite patterns in their tumors to identify cancer-related pathway that are impacted by diabetes. This study is ongoing and will assess whether type 1 and 2 diabetes induce changes to tumor biology that enhance the odds of disease progression. We are particularly interested in changes to metabolic pathways, and how they can be targeted to decrease the negative impact that a diabetes diagnosis may have on breast cancer outcomes.