SignificanceNitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) are inflammation-associated enzymes with oncogenic function in breast cancer. We show that crosstalk between NOS2/COX2 promotes aggressive phenotypes and that elevated coexpression of NOS2/COX2 in tumors predict significantly reduced patient survival (33%) when compared with 95% survival of estrogen receptor-negative patients with low NOS2/COX2 tumor expression. In addition, we have identified a tumor subtype-specific mechanism showing involvement of TNFα and/or endoplasmic reticulum stress as key players in this autocrine loop. Importantly, the simultaneous inhibition of NOS2/COX2 significantly reduced tumor growth in a xenograft murine model, suggesting that targeted inhibition of NOS2/COX2 may be therapeutically beneficial.

Inflammation is implicated in triple negative breast cancer (TNBC) progression. TNBC carries a worse prognosis than other breast cancer subtypes, and with the clinical and molecular heterogeneity of TNBC, there is a lack of effective therapeutic targets available. Identification of molecular targets for TNBC subtypes is crucial towards personalized patient stratification. Inducible nitric oxide synthase (iNOS) has been shown to induce p53 mutation accumulation, basal-like gene signature enrichment and transactivation of the epidermal growth factor receptor (EGFR) via s-nitrosylation. Herein we report that iNOS is associated with disease recurrence, distant metastasis and decreased breast cancer specific survival in 209 cases of TNBC. Employing TNBC cell lines representing normal basal breast, and basal-like 1 and basal-like 2 tumors, we demonstrate that nitric oxide (NO) induces EGFR-dependent ERK phosphorylation in basal-like TNBC cell lines. Moreover NO mediated cell migration and cell invasion was found to be dependent on EGFR and ERK activation particularly in basal-like 2 TBNC cells. This occurred in conjunction with NF-κB activation and increased secretion of pro-inflammatory cytokines IL-8, IL-1β and TNF-α. This provides substantial evidence for EGFR as a therapeutic target to be taken into consideration in the treatment of a specific subset of basal-like TNBC overexpressing iNOS.

Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and constitute promising targets for cancer treatment. Here, we demonstrate that the glucose transporter 4 (GLUT4) has a prominent role in basal glucose uptake in MCF7 and MDA-MB-231 breast cancer cells. We show that shRNA-mediated down-regulation of GLUT4 diminishes glucose uptake and induces metabolic reprogramming by reallocating metabolic flux to oxidative phosphorylation. This reallocation is reflected on an increased activity of the mitochondrial oxidation of pyruvate and lower lactate release. Altogether, GLUT4 inhibition compromises cell proliferation and critically affects cell viability under hypoxic conditions, providing proof-of-principle for the feasibility of using pharmacological approaches to inhibit GLUT4 in order to induce metabolic reprogramming in vivo in breast cancer models.

The relationship between estrogen and some types of breast cancer has been clearly established. However, although several studies have demonstrated the relationship between estrogen and glucose uptake via phosphatidylinositol 3-kinase (PI3K)/Akt in other tissues, not too much is known about the possible cross talk between them for development and maintenance of breast cancer. This study was designed to test the rapid effects of 17β-estradiol (E2) or its membrane-impermeable form conjugated with BSA (E2BSA) on glucose uptake in a positive estrogen receptor (ER) breast cancer cell line, through the possible relationship between key components of the PI3K/Akt signaling pathway and acute steroid treatment. MCF-7 human breast cancer cells were cultured in standard conditions. Then 10 nM E2 or E2BSA conjugated were administered before obtaining the cell lysates. To study the glucose uptake, the glucose fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose was used. We report an ER-dependent activation of some of the key steps of the PI3K/Akt signaling pathway cascade that leads cells to improve some mechanisms that finally increase glucose uptake capacity. Our data suggest that both E2 and E2BSA enhance the entrance of the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, and also activates PI3K/Akt signaling pathway, leading to translocation of glucose transporter 4 to the plasma membrane in an ERα-dependent manner. E2 enhances ER-dependent rapid signaling triggered, partially in the plasma membrane, allowing ERα-positive MCF-7 breast cancer cells to increase glucose uptake, which could be essential to meet the energy demands of the high rate of proliferation.

28Bifidobacterium animalis subsp. lactis IPLA R1 and Bifidobacterium longum IPLA 29 E44 strains were tested in vivo for their safety and ability to modulate the intestinal 30 microbiota. Chemically simulated gastrointestinal digestion showed considerably lower 31 survival of E44 than R1 strain, the first microorganism also being more sensitive to 32 refrigerated storage in 10%-skimmed milk at 4ºC. Harmful glycosidic activities were absent, 33 or at low levels, in the strains R1 and E44. Both strains were sensitive to most antibiotics and 34 resistant to aminoglycosides, a common feature in bifidobacteria. Similarly to several other 35 bifidobacteria strains, B. animalis subsp. lactis IPLA R1 displayed a moderate resistance 36 against tetracycline which correlated with the presence of tet(W) gene in its genome. The 37 general parameters indicating well-being status, as well as translocation to different organs 38 and histological examination of the gut tissues, revealed no changes induced by the 39 administration of bifidobacteria. 12 week-old male Wistar rats were distributed into three 40 groups, eight rats in each. Two groups were administered daily over 10 8 cfu of the 41 corresponding strain suspended in 10%-skimmed milk for 24 days, whereas rats in the 42 placebo group received skimmed milk without microorganisms added. The microbiota and 43 short chain fatty acids (SCFA) were monitored in faeces at different time points during 44 treatment and in caecum-content at the end of the assay. Quantitative PCR (qPCR) showed 45 that faecal and caecal Bifidobacterium levels were higher in bifidobacteria-fed rats than in the 46 placebo rats at the end of the intervention, whereas total anaerobic-plate counts did not show 47 significant differences. Quantification of B. animalis and B. longum by qPCR showed that, 48 independent of the microorganism administered, treatment with bifidobacteria resulted in 49 higher levels of B. animalis in the caecum. PCR-DGGE analysis of microbial populations 50 revealed a higher diversity of bands in caecum-content of rats fed B. animalis IPLA R1 than 51 in the placebo group and rats fed B. longum IPLA E44. Remarkably, although no variations in 52 3 the proportion of acetate, propionate and butyrate were found, at the end of the assay the total 53 SCFA concentration in the faeces of rats fed bifidobacteria was significantly higher and those 54 in caecum-content significantly lower, than that of the placebo group. This suggests a 55 displacement of the SCFA production to parts of the colon beyond the caecum in rats 56 receiving bifidobacteria. Therefore, the oral administration of B. animalis IPLA R1 and B. 57 longum E44 can be considered safe, these microorganisms having the ability to modulate the 58 intestinal microbiota of rats by influencing SCFA and the bifidobacterial population levels.