Novel Markers for Disease Outcome in Breast Cancer

项目1：我们继续对非裔美国人和欧裔美国人er阳性和er阴性乳腺癌患者的代谢组学、蛋白质组学和转录组学进行综合检测，以发现生物标志物。这项研究的前景是发现新的预后生物标志物，并阐明可能导致非裔美国女性乳腺癌侵袭性的原因。使用非靶向发现方法和关键代谢物的验证，我们表征了人类乳腺肿瘤的代谢组学特征，并揭示了这些肿瘤的内在代谢物特征。重要的是，肿瘤代谢物2-羟基戊二酸（2HG）在肿瘤和人类乳腺癌细胞系的一个亚群中积累。2HG达到了与异柠檬酸脱氢酶（IDH）突变胶质瘤相当的摩尔浓度，尽管没有IDH突变。相反，我们发现2HG水平升高与乳腺癌中MYC通路激活之间存在显著关联，这在人类乳腺上皮细胞和诱导MYC过表达和敲低的乳腺癌细胞中得到了证实。进一步的分析显示，在2HG-高的肿瘤中，DNA甲基化在全球范围内增加，并确定了一种生存率较低的肿瘤亚型，具有明显的DNA甲基化，高组织2HG，并且在非裔美国患者中发生率较高。该亚型肿瘤具有WNT和MYC通路激活的干细胞样转录特征。这些肿瘤过度表达谷氨酰胺酶，提示乳腺癌中谷氨酰胺与2HG代谢之间存在功能关系。因此，13c标记的谷氨酰胺在2HG异常积累的细胞中代谢为2HG，而药理学和sirna介导的谷氨酰胺酶抑制显著降低2HG。我们的研究结果强调2HG是与MYC激活和不良预后相关的候选乳腺癌肿瘤代谢物。这些研究仍在继续，我们目前正在评估2HG的致癌作用，以及它们与ADHFE1表达的关系，ADHFE1是一种产生2HG的线粒体酶。目前的数据显示，2HG是一种乳腺癌肿瘤代谢物，可增加人乳腺癌细胞的干细胞样和转移特征，而过表达ADHFE1可诱导相同的表型，并增强癌细胞中的还原性羧化，导致2HG增加。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。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）、环氧合酶-2和胱硫氨酸合酶是炎症标志物，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。我们实验室最近的研究得出了一项新颖且具有临床意义的观察结果，即NOS2表达与预后基底样转录模式相关，并且是雌激素受体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。此外，我们发现这些NOS2诱导的刺激物上调了侵袭性癌症表型的关键指标，包括S100钙结合蛋白A8、IL-6、IL-8和组织抑制剂基质金属蛋白酶-1，而MDA-MB-231乳腺癌细胞中NOS2的抑制抑制了相同的标志物。NO还改变了MDA-MB-231细胞对紫杉醇和其他化疗药物的细胞迁移和化疗耐药性。最值得注意的是，在裸鼠中，当NOS2被抑制时，MDA-MB-231肿瘤异种移植物的生长和脑转移明显受到抑制。这些新结果进一步将NOS2升高与癌症进展联系起来，并表明NO的产生调节乳腺癌细胞的化疗耐药和转移。有了这些观察，我们最近开始评估半胱硫氨酸合酶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，半胱硫氨酸是半胱硫氨酸-合成酶（CBS）的产物，可在乳腺肿瘤中积累。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，以及它们如何能够减少糖尿病诊断对乳腺癌结果的负面影响。

Project 1: We continued to comprehensively examine the metabolome, proteome and transcriptome of ER-positive and ER-negative breast tumors from African-American and European-American patients for biomarker discovery. The promise of the study is the discovery of novel biomarkers for prognosis, and for elucidating what may drive the aggressiveness of breast cancer in African-American women. Using an untargeted discovery approach and validation of key metabolites, we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors. Importantly, the oncometabolite, 2-hydroxyglutarate (2HG), accumulated in a subset of tumors and human breast cancer cell lines. 2HG reached mmolar concentrations comparable to those in isocitrate dehydrogenase (IDH)-mutant gliomas, despite the absence of IDH mutations. Instead, we discovered a significant association between increased 2HG levels and MYC pathway activation in breast cancer, which was corroborated in human mammary epithelial and breast cancer cells with inducible MYC overexpression and knockdown. Further analyses showed a global increase of DNA methylation in 2HG-high tumors and identified a poor survival tumor subtype with distinct DNA methylation, high tissue 2HG, and heightened occurrence in African-American patients. Tumors of this subtype had a stem cell-like transcriptional signature with WNT and MYC pathway activation. These tumors over-expressed glutaminase, suggesting a functional relationship between glutamine and 2HG metabolism in breast cancer. Accordingly, 13C-labeled glutamine was metabolized into 2HG in cells with aberrant 2HG accumulation, whereas pharmacologic and siRNA-mediated inhibition of glutaminase markedly reduced 2HG. Our findings highlight 2HG as a candidate breast cancer oncometabolite associated with MYC activation and poor prognosis. These studies are being continued, and we are currently evaluating the oncogenic effects of 2HG and how they relate to the expression of ADHFE1, a mitochondrial enzyme that produces 2HG. Current data show that 2HG is a breast cancer oncometabolite that increases stem cell-like and metastatic features of human breast cancer cells while overexpression of ADHFE1 induces the same phenotypes and enhances reductive carboxylation in cancer cells, leading to increased 2HG. 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. Project 2: Inflammation is widely recognized as an inducer of cancer progression. Inducible nitric oxide synthase (NOS2), cyclooxygenase-2 and cystathionine beta synthase are 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. Recent 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. Moreover, we found that key indicators of an aggressive cancer phenotype including increased S100 calcium binding protein A8, IL-6, IL-8, and tissue inhibitor matrix metalloproteinase-1 are up-regulated by these NOS2-induced stimulants, whereas inhibition of NOS2 in MDA-MB-231 breast cancer cells suppressed the same markers. NO also altered cellular migration and chemoresistance of MDA-MB-231 cells to Taxol and other chemotherapeutics. Most notably, MDA-MB-231 tumor xenograft growth and metastases to the brain were significantly suppressed when NOS2 was inhibited in nude mice. These novel results further link elevated NOS2 to cancer progression and show that NO production regulates chemoresistance and metastasis of breast cancer cells. Having made these observations, we recently, started to evaluate the role of cystathionine beta synthase 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 cystathionine beta synthase (CBS), accumulates in breast 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 signatures 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 particular 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.