Novel Markers for Disease Outcome in Breast Cancer

乳腺癌疾病结果的新标志物

• 批准号: 8937885
• 负责人: Stefan Ambs
• 金额: $ 59.5万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937885
• 关键词: Affect; African American; Age; American; Area; Baltimore; Biochemical Pathway; Biological; Biological Markers; Blood specimen; Brain; Breast Cancer Cell; Calgranulin A; Cancer Biology; Cancer Patient; Cancer Research Project; Cancer cell line; Categories; Cells; Characteristics; Clinical Research; Collaborations; Consent; Cystathionine; Cystathionine beta-Synthase; DNA Methylation; DNA Modification Process; Data; Data Set; Development; Disease; Disease Marker; Disease Outcome; Disease Progression; Education; Enrollment; Enzymes; Epithelial; Estrogen receptor negative; Estrogen receptor positive; Ethnic Origin; European; Event; Fatty acid glycerol esters; Freezing; Frequencies; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic; Genetic Transcription; Genetic Variation; Glioma; Glutaminase; Glutamine; Goals; Household; Human; Hydrogen Sulfide; Hypoxia; Income; Inflammation; Interferon Type II; Interleukin-6; Interstitial Collagenase; Isocitrate Dehydrogenase; Label; Laboratories; Life; Link; Lipids; Logistic Regressions; Low income; MDA MB 231; Malignant Neoplasms; Mammary Gland Parenchyma; Mammary Neoplasms; Mammary gland; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Medicine; Metabolic; Metabolism; Methods; Molecular; Mutation; NOS2A gene; Neoplasm Metastasis; Nitric Oxide; Nude Mice; Outcome; Oxidation-Reduction; Paclitaxel; Pathway interactions; Patient Self-Report; Patients; Pattern; Phenotype; Pilot Projects; Production; Proteins; Proteome; Race; Regression Analysis; Regulation; Research; Resistance; Ribonucleotide Reductase Subunit Gene; Role; Schedule; Serum; Signaling Molecule; Small Interfering RNA; Social isolation; Socioeconomic Status; Specimen; Stem cells; Stress; Stressful Event; Surveys; Tamoxifen; Testing; Tissue Microarray; Tissue Sample; Tissues; Tobacco smoking; Tumor Biology; Tumor Markers; Tumor Subtype; United States; Up-Regulation; Validation; Winking; Withdrawal; Woman; Work; Wound Healing; angiogenesis; base; cancer surgery; carcinogenesis; cdc Genes; cell motility; clinically significant; college; cyclooxygenase 2; design; experience; feeding; health disparity; human NOS2A protein; inflammatory marker; inhibitor/antagonist; malignant breast neoplasm; metabolomics; mortality; mutant; novel; novel marker; novel strategies; outcome forecast; overexpression; prognostic; pyrimidine metabolism; response; ribonucleotide reductase M2; trend; tumor; tumor microenvironment; tumor progression; tumor xenograft

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.

项目1：炎症被广泛认为是癌症进展的诱导剂。诱导型一氧化氮合酶（NOS2），环氧合酶-2和胱硫唑β-合酶是炎症标记，参与伤口愈合，血管生成和致癌作用。 NOS2上调和一氧化氮（NO）产生增加还会影响细胞的氧化还原状态，并诱导蛋白质，脂质和DNA修饰。我们实验室的最新研究导致了新颖且临床上显着的观察结果，即NOS2表达与预后的基础样转录模式有关，并且是雌激素受体（ER）阴性乳腺肿瘤女性生存不良的独立预测指标。目前，这些发现与NCI的David Wink博士实验室合作进一步追求。在2013财年，这种合作表明，在ER阴性乳腺癌细胞中NOS2的上调是响应缺氧，血清戒断，干扰素伽马和外源性NO的响应，与乳腺癌生物学中肿瘤微环境对NO产生的NO生产的调节一致。此外，我们发现，包括S100钙结合蛋白A8，白介素-6和-8以及组织抑制剂基质金属蛋白酶-1的主要指标，这些NOS2诱导的刺激剂在MDA-MB-231乳腺癌细胞中对NOS2的抑制作用均被这些NOS2诱导的刺激剂上调。也没有改变MDA-MB-231细胞对紫杉醇和其他化学治疗药的细胞迁移和化学抗性。最值得注意的是，当Nude小鼠中NOS2抑制NOS2时，MDA-MB-231肿瘤异种移植物和细胞转移从脂肪垫向大脑显着抑制。这些新的结果进一步将NOS2升高到癌症的进展，并表明没有生产调节乳腺癌细胞的化学抗性和转移。在做出这些观察结果之后，我们最近开始评估胱硫唑β合酶在乳腺癌进展中的作用。这种酶像NOS2一样，释放出硫化氢的气态信号分子。硫化氢（NO）刺激血管生成。初步数据表明，胱淀粉β-合酶的产物Cystathionine积聚在乳腺肿瘤中。项目2：我们继续全面研究来自非裔美国人和欧美裔美国人的生物标志物发现的ER阳性和ER阴性乳腺肿瘤的代谢组，蛋白质组和转录组。该研究的希望是发现了预后的新型生物标志物，并阐明了可能推动非裔美国妇女乳腺癌侵略性的原因。我们使用未靶向的发现方法和关键代谢产物的验证，表征了人类乳腺肿瘤的代谢组谱，并在这些肿瘤中发现了内在的代谢产物特征。重要的是，在肿瘤和人类乳腺癌细胞系中积累了oncometabolite，2-羟基戊二酸（2HG）。尽管没有IDH突变，但2HG达到了与异氯酸酯脱氢酶（IDH） - 突变胶质瘤相当的Mmolol浓度。取而代之的是，我们发现乳腺癌的2HG水平升高与MYC途径激活之间存在显着关联，这在人类乳腺上皮和乳腺癌细胞中得到了证实，具有诱导的MYC过表达和敲低。进一步的分析表明，在2HG高肿瘤中，DNA甲基化的全球增加，并鉴定出较差的生存率肿瘤亚型，具有明显的DNA甲基化，高组织2HG，并且在非裔美国人患者中发生了增强。该亚型的肿瘤具有带有Wnt和MYC途径激活的干细胞样转录特征。这些肿瘤过表达的谷氨酰胺酶，表明乳腺癌中谷氨酰胺与2HG代谢之间存在功能关系。因此，将13C标记的谷氨酰胺代谢为异常2Hg积累的细胞2HG，而药理学和siRNA介导的谷氨酰胺酶的抑制显着降低了2HG。我们的发现突出显示了2HG作为候选乳腺癌oncometabolite与MYC激活和预后不良相关的乳腺癌。通过与贝勒医学院的Sreekumar实验室合作，我们还使用质谱测量了腔和基础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物化学途径联系起来，并开发了一种新型的基于基于等级的方法，以根据患者的富集途径在患者播放的途径中选择途径。然后，该途径的主要介体因其在乳腺癌进展中的作用而表征。我们发现，在乳腺癌中，嘧啶的代谢通常会改变，并且一个相关的关键基因，核糖核苷酸还原酶亚基M2（RRM2）预测，所有乳腺癌亚型的存活率降低，以及在抗他莫昔芬的腔内患者中的生存率降低。使用组织微阵列验证了他莫昔芬耐药患者的RRM2表达增加，而在他莫昔芬耐药细胞和异种移植肿瘤中，RRM2的代谢产物较高。该关键酶在他莫昔芬耐药细胞中遗传学和药理抑制都显着降低了增殖，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。这项研究提出，将与RRM2相关的代谢产物评估为他莫昔芬耐药性的非侵入性标记及其药理抑制作用，作为一种克服他莫昔芬在乳腺癌中耐药性的新方法。项目3：我们启动了一个评估压力性生活事件对肿瘤生物学的影响的项目。在一项临床研究中，我们将为计划进行乳腺癌手术的乳腺癌患者提供简短的调查，以评估他们的压力和社会隔离。我们还将从这些患者那里收集冷冻的肿瘤和邻近的正常乳房组织以及血液样本，并评估乳腺组织或血液样本是否具有与其感知的压力和社会隔离状态有关的生物学特征。我们假设，感知到高的压力暴露的患者具有与更具侵略性疾病和较差生存率一致的生物学特征。该试点研究旨在从同意的患者中收集100对肿瘤/正常对，并完成了完整的调查。目前，我们已经招募了38名患者，并从其中25例收集了新鲜的肿瘤标本。在这种情况下，我们还评估了乳腺肿瘤中社会经济状况与p53突变的发生之间的关系。我们分析了来自美国大巴尔的摩地区的173名乳腺癌患者，分析了肿瘤p53突变状态与肿瘤特征，教育和自我报告的年收入（HI）的关联。结果：p53突变频率与HI显着相关。 15,000美元HI的患者的P53突变频率最高（21％），其次是收入组15,000至60,000美元（18％），而高于60,000美元的HI的患者的突变最少（5％）。当二分法为60,000美元时，低收入类别的135名患者中有26个获得了P53突变，而38例高收入中只有2个具有突变。在具有3个收入类别的调整后的逻辑回归分析（趋势测试）中，HI和p53突变状态之间的关联与肿瘤特征，年龄，种族/种族，烟草吸烟和体重无关。进一步的分析表明，HI可能会在发展雌激素受体（ER）阴性疾病的患者中优先影响p53突变频率。结论：HI与患有ER阴性疾病的患者的p53突变频率有关。