Novel Markers for Disease Outcome in Breast Cancer

项目1：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发现，NOS2表达与预后基底样转录模式相关，是雌激素受体（ER）阴性乳腺肿瘤患者生存不良的独立预测因子。这些发现目前正在与NCI的David Wink博士的实验室合作进行进一步的研究。2013年，该合作研究表明，在er阴性乳腺癌细胞中，NOS2的上调发生在缺氧、血清停药、干扰素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、白细胞介素-6和-8以及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，在裸小鼠中，当NOS2被抑制时，MDA-MB-231肿瘤异种移植和细胞从脂肪垫向脑转移被显著抑制。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。初步数据表明，半胱硫氨酸是半胱硫氨酸合酶的产物，可在乳腺肿瘤中积累。项目2：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。与贝勒医学院的Sreekumar实验室合作，我们还使用质谱法测量了管腔和基底样乳腺癌细胞系的代谢组，并使用基因集分析将这些代谢物与生化途径联系起来，并开发了一种新的基于排序的方法，根据患者来源的组学数据集和预后相关性来选择途径。该通路的关键介质随后被描述为它们在乳腺癌进展中的作用。我们发现，嘧啶代谢在乳腺癌中普遍发生改变，一个相关的关键基因，核糖核苷酸还原酶亚基M2 (RRM2)，预测所有乳腺癌亚型以及对他莫昔芬耐药的luminal患者的生存率降低。使用组织微阵列技术证实了RRM2在他莫昔芬耐药患者中的表达增加，而RRM2的代谢产物在他莫昔芬耐药细胞和异种移植肿瘤中更高。在他莫昔芬耐药细胞中，该关键酶的遗传和药理学抑制均显著降低了细胞增殖，降低了细胞周期基因的表达，并使细胞对他莫昔芬治疗敏感。本研究建议评估rrm2相关代谢物作为他莫昔芬耐药的无创标记物及其药理抑制作为克服乳腺癌他莫昔芬耐药的新方法。项目3：我们开始了一个评估生活压力事件对肿瘤生物学影响的项目。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。目前，我们已经招募了38名患者，并从其中25名患者中收集了新鲜冷冻肿瘤标本。在此背景下，我们还评估了社会经济地位与乳腺肿瘤中p53突变（预后不良的标志）发生之间的关系。我们分析了来自美国大巴尔的摩地区的173名乳腺癌患者的肿瘤p53突变状态与肿瘤特征、教育程度和自我报告的家庭年收入（HI）的关系。结果：p53突变频率与HI有显著相关性。HI值为1.5万美元的患者p53突变频率最高（21%），其次是收入在1.5万美元至6万美元之间的人群（18%），而HI值高于6万美元的人群p53突变频率最少（5%）。在6万美元的二分法中，135名低收入组患者中有26人获得了p53突变，而38名高收入组患者中只有2人携带突变。在3个收入类别（趋势检验）的调整logistic回归分析中，HI与p53突变状态的相关性与肿瘤特征、年龄、种族/民族、吸烟和体重无关。进一步分析表明，HI可能优先影响雌激素受体（ER）阴性疾病患者的p53突变频率。结论：在er阴性疾病患者中，HI与p53突变频率相关。

Project 1: 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 estrogen receptor (ER)-negative breast tumors. These findings are currently further pursued in collaboration with the laboratory of Dr. David Wink at the NCI. In FY13, this collaboration showed that up-regulation of NOS2 in ER-negative breast cancer cells occurs in response to hypoxia, serum withdrawal, interferon 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, interleukin-6 and -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 xenografts and cell metastases from the fat pad to the brain were significantly suppressed when NOS2 was inhibited in the 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. Preliminary data show that cystathionine, a product of cystathionine beta synthase, accumulates in breast tumors. Project 2: 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. In collaboration with the Sreekumar laboratory at Baylor College of Medicine, we also measured the metabolome of luminal and basal-like breast cancer cell lines using mass spectrometry and linked these metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in breast cancer progression. We found that pyrimidine metabolism was commonly altered in breast cancer, and one associated key gene, ribonucleotide reductase subunit M2 (RRM2), predicted decreased survival across all breast cancer subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. This study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in breast cancer. Project 3: We started a project evaluating the impact of stressful life events 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. Currently, we have enrolled 38 patients and have collected fresh-frozen tumors specimens from 25 of those. In this context, we also evaluated the relationship between socioeconomic status and the occurrence of a p53 mutation, a marker of poor outcome, in breast tumors. We analyzed the association of the tumor p53 mutational status with tumor characteristics, education, and self-reported annual household income (HI) among 173 breast cancer patients from the greater Baltimore area, United States. Results: p53 mutational frequency was significantly associated with HI. Patients with $15,000 HI had the highest p53 mutation frequency (21%), followed by the income group between $15,000 and $60,000 (18%), while those above $60,000 HI had the fewest mutations (5%). When dichotomized at $60,000, 26 out of 135 patients in the low income category had acquired a p53 mutation, while only 2 out of 38 with a high income carried a mutation. In the adjusted logistic regression analysis with 3 income categories (trend test), the association between HI and p53 mutational status was independent of tumor characteristics, age, race/ethnicity, tobacco smoking and body mass. Further analyses showed that HI may impact the p53 mutational frequency preferentially in patients who develop an estrogen receptor (ER)-negative disease. Conclusions: HI is associated with the p53 mutational frequency in patients who develop an ER-negative disease.