Project 1: Inflammation is widely recognized as an inducer of cancer progression. Inducible nitric oxide synthase (NOS2), cyclooxygenase-2 and cystathionine beta synthase (CBS) 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 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 xenografts and cell metastases from the fat pad 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 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. 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 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. These studies are being continued and we are currently evaluating the oncogenic effects of 2HG and compare those to the effects of ADHFE1, a mitochondrial enzyme that produces 2HG. 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 65 patients and have collected fresh-frozen tumors specimens from 36 of those.
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