Project 1: 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 previously 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. We focused our investigations on ADHFE1 because ADHFE1 and MYC locus amplifications co-occur in breast tumors. Additional data indicated the existence of a regulatory feedback loop between c-Myc signaling and ADHFE1 protein expression. 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. Additional research is needed to elucidate the potentially oncogenic function of 4HB synthesis in breast cancer 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. 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 alsoaffects 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. 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 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.
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