Exploring Molecular Links between Obesity and Breast Cancer

Obesity is associated with a high risk of incidence of, and mortality for, postmenopausal breast cancer. Despite this well-established link, the molecular and mechanistic basis of the obesity and breast cancer association still remains unclear. In obesity research, genetic variation due to copy number differences has become increasingly popular. The salivary amylase gene, AMY1, is well-known for its extensive copy number variation (CNV) in the human genome and has previously been correlated with a genetic predisposition toward obesity; however, research surrounding this association is controversial. Despite an established relationship between obesity and breast cancer risk, the recently reported genetic association between AMY1 CNV and obesity has not yet been examined in normal and obese breast cancer patients. Furthermore, gene expression changes in breast tumours from obese women remain poorly characterised. We hypothesise that obese breast cancer patients are associated with (1) low AMY1 copy number and (2) differential expression of candidate genes in the breast tumour. This study included 55 post-menopausal breast cancer patients from The Cancer Society Tissue Bank, with a BMI (body mass index)> 30 (obese; n=28) or BMI < 25 (healthy; n=27). Quantitative PCR (qPCR) assessment of germline AMY1 copy number status from blood showed that obese breast cancer patients have a lower average copy number of AMY1 compared to normal weight patients. Examining breast tumour expression profiles of obese and non-obese patients from two published studies, identified four candidate genes (GRIA2, DUSP4, NR2F1, and ADH1B) shared between both studies. Analysis of gene expression data from The Cancer Genome Atlas (TCGA) indicated that these four genes are differentially expressed within clinically relevant breast tumour subtypes characterised by oestrogen receptor, progesterone receptor and HER2 status. qPCR analysis of each candidate gene within our study cohort showed that the average expression of GRIA2, DUSP4, NR2F1 and ADH1B was lower in obese compared to healthy breast tumours, but these results were not statistically significant. My study indicated that BMI may be associated with lower germline copy number of AMY1 in post-menopausal breast cancer patients; however, further work with a larger cohort is needed to establish if GRIA2, DUSP4, NR2F1 and ADH1B are associated with obesity related breast cancer

Local and systemic effects of adipocyte-secreted factors in breast cancer

Breast cancer is a complex disease that, once developed, progresses in response to multiple environmental factors, including local microenvironmental factors within the breast and systemic markers in circulation. Obesity affects one third of all New Zealand adults and is known to negatively impact breast cancer outcomes. Epidemiological studies have shown obese women with breast cancer have increased risk of recurrence and metastasis, poorer pathological response rates to chemotherapy, and worse overall survival. The biological mechanisms underlying these associations are complex and not yet completely understood. Cancer associated adipocytes (CAA) are fat cells located within close proximity to breast tumour cells. In vitro, CAA promote breast cancer cell migration, invasion, and resistance to therapy. Analysis of gene expression in breast cancer cells co-cultured with CAA has identified a number of genes which may be supporting disease progression. To further assess the influence of CAA on breast cancer cells, we identified and quantified changes in global protein abundance induced in breast cancer cells co-cultured with human breast adipocytes (CAA), and evaluated these changes by identifying key molecules and pathways that were significantly altered. Global differences in relative protein expression in MCF-7 (ER+, PR+, HER2-) and MDA-MB-231 (ER-, PR-, HER2-) breast cancer cells co-cultured with, or without, mature breast adipocytes in a transwell co-culture system, were measured using isobaric tags for relative and absolute quantification (iTRAQ) labelling and liquid chromatography tandem mass spectrometry (LC-MS/MS). In both control and co-cultured samples, a total of 1,126 proteins and 1,218 proteins were identified in MCF-7 and MDA-MB-231 breast cancer cells, respectively. Relative to controls, 85 proteins in MCF-7 cells (32 upregulated, 53 downregulated) and 63 proteins in MDA-MB-231 cells (51 upregulated, 12 downregulated) were differentially abundant by 1.5-fold or greater in co-cultured cells. Co-culture with CAA caused an enriched upregulation of tricarboxylic acid (TCA) cycle proteins in MCF-7 cells and glycolysis proteins in MDA-MB-231 cells. The glycolytic protein, phosphoglycerate kinase 1 (PGK1), was the only protein that was upregulated by more than 1.5-fold in both MCF-7 and MDA-MB-231 cells co-cultured with CAA. PGK1 is a kinase enzyme that plays an important role in the glycolytic pathway. In women with breast cancer, increased PGK1 expression in the tumour has been identified as a predictor of poor patient survival and marker of resistance to paclitaxel. As metabolic co-operation between adipocytes and breast cancer cells is a key mechanism promoting breast tumour progression, we investigated PGK1 overexpression in vitro. The transient transfection model for in vitro PGK1 overexpression utilised in this study induced differential effects in MCF-7 and MDA-MB-231 breast cancer cells. PGK1 overexpression increased sensitivity to chemotherapy in MCF-7 cells. Whereas, cell proliferation and viability were decreased, and conditioned media lactate concentrations were increased, in GFP and PGK1 expressing plasmid transfected MDA-MB-231 cells. In silico analysis showed PGK1 expression was higher in HER2 enriched compared to triple negative breast cancer cells, and was upregulated in HER2 overexpressing (HER2+) compared to HER2- breast tumours, suggesting that PGK1 expression may be particularly relevant to HER2+ breast cancers. Obesity is characterised by a state of low-grade chronic systemic inflammation. Breast cancer chemotherapies are predominantly metabolised in liver hepatocytes by cytochrome P450 (CYP) drug metabolising enzymes. Inflammatory cytokines have been shown to downregulate expression and activity of CYP enzymes in vitro. Additionally, CYP genotype-phenotype discordance has been observed in patients with advanced cancer. To investigate whether obesity-associated circulating inflammatory cytokines influence in vivo activity of CYP enzymes in women receiving chemotherapy for breast cancer, we carried out an exploratory patient study that recruited seven non-obese and five obese women receiving adriamycin-cyclophosphamide (AC) and paclitaxel chemotherapy for stage II or III breast cancer. During chemotherapy, serum levels of B-cell activating factor (BAFF), growth and differentiation factor 15 (GDF-15) and monocyte chemoattractant protein 1 (MCP-1) increased, whereas interleukin 10 (IL-10) levels decreased. Importantly, changes in the levels of circulating inflammatory cytokines during chemotherapy were not associated with differences in body morphometry or voluntary physical activity levels. Activity of the CYP enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4), measured using the ‘Inje’ probe drug cocktail, were largely unchanged over the course of chemotherapy, although varied between participants. However, increased serum MCP-1 levels correlated with decreased CYP3A4 activity during chemotherapy, and this finding provides preliminary evidence that circulating inflammatory cytokines may negatively influence CYP-mediated chemotherapy metabolism in women undergoing treatment for breast cancer. This study has provided, for the first time, an extensive list of breast cancer cell protein abundance alterations induced by co-culture with CAA, which can be used as a comprehensive platform for future investigations. Moreover, this study has validated, for the first time, the feasibility of using the ‘Inje’ cocktail to measure CYP activity in women receiving chemotherapy for breast cancer, and in doing so, has provided preliminary evidence to support the concept that changes in circulating inflammatory cytokines during chemotherapy treatment may impact CYP activity, and thus, chemotherapy metabolism in some patients

Characterisation of methylglyoxal stress in human colorectal cancer and liver metastases using immunohistochemistry.

Background: Glycolysis is the principal source of energy for cancerous cells. One inevitable consequence of the elevated glycolytic rate is the production of highly reactive molecules such as methylglyoxal (MG). MG induces the glycation of proteins on lysine and arginine residues and generates protein adducts called MG-derived hydroimidazolones (MGHs). Glyoxalase 1 (GLO1) is the main detoxifying enzyme of MG. It is expressed in most eukaryotes and prokaryotes and is localized in the cytoplasmic compartment. An increase of GLO1 expression and activity is a cell defence mechanism against glycation damage induced under MG stress. Our previous studies reported the presence of MG protein adducts in CRC tumours and have linked MG stress with the resistance to targeted therapy in KRAS-mutated CRCs. Aims: In this pilot project, we undertook the detection of MG stress in human CRC primary tumours and liver metastases lesions. Methods: We have used immunohistochemistry and antibodies directed against MGHs protein adducts and GLO1 enzyme in CRC samples. Specific Ki67 antibodies were used for the evaluation of tumour proliferation rate. Results: By comparison of the same histological sample for GLO1 and Ki67 immunostainings, we observed that GLO1 enzyme was strongly detectable in the nucleus of undifferentiated and highly proliferative human CRC lesions. While most of the well-differentiated CRC tumours demonstrated undetectable to low nuclear GLO1 levels in the nucleus. Cytoplasmic GLO1 was similarly distributed among differentiated and non-differentiated tumours. Conclusion: It might be therefore interesting to explore further this peculiar GLO1 sub-localisation that could potentially indicate for the first time the presence of MG stress in the nucleus and the necessity for the nuclear translocation of GLO1 detoxifying enzyme in aggressive CRC lesions. Whether nuclear GLO1 detection could be a valuable marker in terms of unfavourable prognosis in CRC patients will be analysed on a large collection of CRC patients with documented clinical data and follow-up

Methylglyoxal: a novel upstream regulator of DNA methylation.

peer reviewed[en] BACKGROUND: Aerobic glycolysis, also known as the Warburg effect, is predominantly upregulated in a variety of solid tumors, including breast cancer. We have previously reported that methylglyoxal (MG), a very reactive by-product of glycolysis, unexpectedly enhanced the metastatic potential in triple negative breast cancer (TNBC) cells. MG and MG-derived glycation products have been associated with various diseases, such as diabetes, neurodegenerative disorders, and cancer. Glyoxalase 1 (GLO1) exerts an anti-glycation defense by detoxifying MG to D-lactate. METHODS: Here, we used our validated model consisting of stable GLO1 depletion to induce MG stress in TNBC cells. Using genome-scale DNA methylation analysis, we report that this condition resulted in DNA hypermethylation in TNBC cells and xenografts. RESULTS: GLO1-depleted breast cancer cells showed elevated expression of DNMT3B methyltransferase and significant loss of metastasis-related tumor suppressor genes, as assessed using integrated analysis of methylome and transcriptome data. Interestingly, MG scavengers revealed to be as potent as typical DNA demethylating agents at triggering the re-expression of representative silenced genes. Importantly, we delineated an epigenomic MG signature that effectively stratified TNBC patients based on survival. CONCLUSION: This study emphasizes the importance of MG oncometabolite, occurring downstream of the Warburg effect, as a novel epigenetic regulator and proposes MG scavengers to reverse altered patterns of gene expression in TNBC

Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response

peer reviewedPancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine. We observed that human PDAC tumors expressing elevated levels of glycolytic enzymes together with high levels of glyoxalase 1 (GLO1), the major MG-detoxifying enzyme, present with a poor prognosis. Next, we showed that glycolysis and subsequent MG stress are triggered in PDAC cells rendered resistant to gemcitabine when compared with parental cells. In fact, acquired resistance, following short and long-term gemcitabine challenges, correlated with the upregulation of GLUT1, LDHA, GLO1, and the accumulation of MG protein adducts. We showed that MG-mediated activation of heat shock response is, at least in part, the molecular mechanism underlying survival in gemcitabine-treated PDAC cells. This novel adverse effect of gemcitabine, i.e., induction of MG stress and HSR activation, is efficiently reversed using potent MG scavengers such as metformin and aminoguanidine. We propose that the MG blockade could be exploited to resensitize resistant PDAC tumors and to improve patient outcomes using gemcitabine therapy

Ascorbate content of clinical glioma tissues is related to tumour grade and to global levels of 5-hydroxymethyl cytosine.

peer reviewedGliomas are incurable brain cancers with poor prognosis, with epigenetic dysregulation being a distinctive feature. 5-hydroxymethylcytosine (5-hmC), an intermediate generated in the demethylation of 5-methylcytosine, is present at reduced levels in glioma tissue compared with normal brain, and that higher levels of 5-hmC are associated with improved patient survival. DNA demethylation is enzymatically driven by the ten-eleven translocation (TET) dioxygenases that require ascorbate as an essential cofactor. There is limited data on ascorbate in gliomas and the relationship between ascorbate and 5-hmC in gliomas has never been reported. Clinical glioma samples (11 low-grade, 26 high-grade) were analysed for ascorbate, global DNA methylation and hydroxymethylation, and methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Low-grade gliomas contained significantly higher levels of ascorbate than high-grade gliomas (p = 0.026). Levels of 5-hmC were significantly higher in low-grade than high-grade glioma (p = 0.0013). There was a strong association between higher ascorbate and higher 5-hmC (p = 0.004). Gliomas with unmethylated and methylated MGMT promoters had similar ascorbate levels (p = 0.96). One mechanism by which epigenetic modifications could occur is through ascorbate-mediated optimisation of TET activity in gliomas. These findings open the door to clinical intervention trials in patients with glioma to provide both mechanistic information and potential avenues for adjuvant ascorbate therapy

Role of MG-stress in PDAC resistance to gemcitabine

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Contribution of methylglyoxal stress to acquired resistance to Gemcitabine in pancreatic ductal adenocarcinoma

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