Modeling Risk Factors for Sleep- and Adiposity-Related Cardiometabolic Disease: Protocol for the Short Sleep Undermines Cardiometabolic Health (SLUMBRx) Observational Study

Background: Obesity and short sleep duration are significant public health issues. Current evidence suggests that these conditions are associated with cardiovascular disease, metabolic syndrome, inflammation, and premature mortality. Increased interest in the potential link between obesity and short sleep duration, and its health consequences, has been driven by the apparent parallel increase in the prevalence of both conditions in recent decades, their overlapping association with cardiometabolic outcomes, and the potential causal connection between the two health issues. The SLUMBRx (Short Sleep Undermines Cardiometabolic Health) study seeks to contribute to the development of a comprehensive adiposity-sleep model while laying the groundwork for a future research program that will be designed to prevent and treat adiposity- and sleep-related cardiometabolic disease risk factors. Objective: This SLUMBRx study aims to address 4 topics pertinent to the adiposity-sleep hypothesis: the relationship between adiposity and sleep duration; sex-based differences in the relationship between adiposity and sleep duration; the influence of adiposity indices and sleep duration on cardiometabolic outcomes; and the role of socioecological factors as effect modifiers in the relationship between adiposity indices, sleep, and cardiometabolic outcomes. Methods: SLUMBRx will employ a large-scale survey (n=1000), recruiting 159 participants (53 normal weight, 53 overweight, and 53 obese) to be assessed in 2 phases. Results: SLUMBRx was funded by the National Institutes of Health, Heart, Lung, and Blood Institute through a K01 grant award mechanism (1K01HL145128-01A1) on July 23, 2019. Institutional Review Board (IRB) approval for the research project was sought and obtained on July 10, 2019. Phase 1 of SLUMBRx, the laboratory-based component of the study, will gather objective adiposity indices (air displacement plethysmography and anthropometrics) and cardiometabolic data (blood pressure, pulse wave velocity and pulse wave analysis, and a blood-based biomarker). Phase 2 of SLUMBRx, a 1-week, home-based component of the study, will gather sleep-related data (home sleep testing or sleep apnea, actigraphy, and sleep diaries). During phase 2, detailed demographic and socioecological data will be collected to contextualize hypothesized adiposity and sleep-associated cardiometabolic disease risk factors. Collection and analyses of these data will yield information necessary to customize future observational and intervention research. Conclusions: Precise implementation of the SLUMBRx protocol promises to provide objective and empirical data on the interaction between body composition and sleep duration. The hypotheses that will be tested by SLUMBRx are important for understanding the pathogenesis of cardiometabolic disease and for developing future public health interventions to prevent its conception and treat its consequences

Heregulin β1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells

Introduction Resistance to anti-epidermal growth factor receptor (anti-EGFR) therapies is an emerging clinical problem. The efficacy of anti-EGFR therapies can be influenced by the presence of heregulins (HRGs), which can bind erbB3/4 receptors and can activate alternative signalling pathways. In the present study we have examined whether HRG signalling can circumvent EGFR blockade in an EGFR-positive tamoxifen-resistant MCF-7 (Tam-R) breast cancer cell line. Methods Tam-R cells, incubated with the selective EGFR tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839), were exposed to HRGβ1 and the effects on erbB receptor dimerization profiles and on activation of associated downstream signalling components were assessed by immunoprecipitation, western blotting and immunocytochemistry. The effects of HRGβ1 on gefitinib-treated Tam-R cell growth and invasion were also examined, and HRGβ1 expression levels were assessed in breast cancer tissue by immunohistochemistry to address the potential clinical relevance of such a resistance mechanism. Results In Tam-R cells, HRGβ1 promoted erbB3/erbB2 and erbB3/EGFR heterodimerization, promoted ERK1/2 and AKT pathway activation and increased cell proliferation and invasion. Gefitinib prevented HRGβ1-driven erbB3/EGFR heterodimerization, ERK1/2 activation and Tam-R cell proliferation, but HRGβ1-driven erbB3/erbB2 heterodimerization, AKT activation and Tam-R cell invasion were maintained. A combination of gefitinib and the phosphatidylinositol 3-kinase inhibitor LY294002 effectively blocked HRGβ1-mediated intracellular signalling activity, growth and invasion in Tam-R cells. Similarly, targeting erbB2 with trastuzumab in combination with gefitinib in Tam-R cells reduced HRGβ1-induced erbB2 and ERK1/2 activity; however, HRGβ1-driven AKT activity and cell growth were maintained while cell invasion was significantly enhanced with this combination. In clinical tissue all samples demonstrated cytoplasmic tumour epithelial HRGβ1 protein staining, with expression correlating with EGFR positivity and activation of both AKT and ERK1/2. Conclusion HRGβ1 can overcome the inhibitory effects of gefitinib on cell growth and invasion in Tam-R cells through promotion of erbB3/erbB2 heterodimerization and activation of the phosphatidylinositol 3-kinase/AKT signalling pathway. This may have implications for the effectiveness of anti-EGFR therapies in breast cancer as HRGβ1 is enriched in many EGFR-positive breast tumours

Endocrine resistance in breast cancer: new roles for ErbB3 and ErbB4

Endocrine resistance is a major limitation to the successful treatment of estrogen receptor-positive (ER+) breast cancer, and the EGFR (epidermal growth factor receptor) and ErbB-2 receptor tyrosine kinases are involved in this process. A recent study now implicates the other two ErbB family members, ErbB-3 and -4. Exposure of ER+ breast cancer cells to the pure antiestrogen, fulvestrant, increased levels of ErbB-3 or ErbB-4 and sensitivity to the growth-stimulatory effects of heregulin ݱ, a potent ligand for these receptors. Thus, the initial growth-inhibitory effects of fulvestrant appear compromised by cellular plasticity that allows rapid compensatory growth stimulation via ErbB-3/4. Further evaluation of pan-ErbB receptor inhibitors in endocrine-resistant disease appears warranted

Polymorphism rs4919510:C>G in Mature Sequence of Human MicroRNA-608 Contributes to the Risk of HER2-Positive Breast Cancer but Not Other Subtypes

BACKGROUND: A few polymorphisms are located in the mature microRNA sequences. Such polymorphisms could directly affect the binding of microRNA to hundreds of target mRNAs. It remains unknown whether rs4919510:C>G located in the mature miR-608 alters breast cancer susceptibility. METHODS: The association of rs4919510:C>G with risk and pathologic features of breast cancer were investigated in two independent case-control studies, the first set including 1,138 sporadic breast cancer patients (including 927 invasive ductal carcinoma patients, 777 of them with known subtypes: 496 luminal-like, 133 HER2-positive, and 148 triple-negative) and 1,434 community-based controls, and the second set including 294 familial/early-onset breast cancer patients and 500 hospital-based cancer-free controls. Odds ratios (ORs) were estimated by logistic regression. Predicted targets of miR-608 and complementary sequences containing rs4919510:C>G were surveyed to reveal potential pathological mechanism. RESULTS: In the first set, although rs4919510:C>G was unrelated to breast cancer in general patients, variant genotypes (CG/GG) were specifically associated with increased risk of HER2-positive subtype (Adjusted OR = 1.97, 95% CI, 1.34-2.90 in the recessive model). Variant G-allele was the risk allele with OR of 1.62 (95% CI, 1.23-2.15). Patients carrying GG-genotype also had larger HER2-positive tumors (P for Kruskal-Wallis test = 0.006). The relationship between rs4919510:C>G and risk of HER2-positive subgroup was validated in the second set (Bonferroni corrected P = 0.06). The adjusted combined OR (total 164 HER2-positive cases) in the recessive model was 1.97 (95% CI, 1.43-2.72) for GG genotype (corrected P = 1.1 × 10(-4)). Bioinformatic analysis indicated that, HSF1, which is required for HER2-induced tumorigenesis, might be a target of miR-608. The minimum free-energy of ancestral-miR-608 (C-allele) binding to HSF1 is -35.9 kcal/mol, while that of variant-form (G-allele) is -31.5 kcal/mol, indicating a lower affinity of variant-miR-608 to HSF1 mRNA. CONCLUSION: rs4919510:C>G in mature miR-608 may influence HER2-positive breast cancer risk and tumor proliferation

Fulvestrant-induced expression of ErbB3 and ErbB4 receptors sensitizes oestrogen receptor-positive breast cancer cells to heregulin β1

Introduction We have previously reported that induction of epidermal growth factor receptor and ErbB2 in response to antihormonal agents may provide an early mechanism to allow breast cancer cells to evade the growth-inhibitory action of such therapies and ultimately drive resistant cell growth. More recently, the other two members of the ErbB receptor family, ErbB3 and ErbB4, have been implicated in antihormone resistance in breast cancer. In the present study, we have investigated whether induction of ErbB3 and/or ErbB4 may provide an alternative resistance mechanism to antihormonal action in a panel of four oestrogen receptor (ER)-positive breast cancer cell lines. Methods MCF-7, T47D, BT474 and MDAMB361 cell lines were exposed to fulvestrant (100 nM) for seven days, and effects on ErbB3/4 expression and signalling, as well as on cell growth, were assessed. Effects of heregulin β1 (HRGβ1) were also examined in the absence and presence of fulvestrant to determine the impact of ER blockade on the capacity of this ErbB3/4 ligand to promote signalling and cell proliferation. Results Fulvestrant potently reduced ER expression and transcriptional activity and significantly inhibited growth in MCF-7, T47D, BT474 and MDAMB361 cells. However, alongside this inhibitory activity, fulvestrant also consistently induced protein expression and activity of ErbB3 in MCF-7 and T47D cells and ErbB4 in BT474 and MDAMB361 cell lines. Consequently, fulvestrant treatment sensitised all cell lines to the actions of the ErbB3/4 ligand HRGβ1 with enhanced ErbB3/4-driven signalling activity, reexpression of cyclin D1 and significant increases in cell proliferation being observed when compared to untreated cells. Indeed, in T47D and MDAMB361 HRGβ1 was converted from a ligand having negligible or suppressive growth activity into one that potently promoted cell proliferation. Consequently, fulvestrant-mediated growth inhibition was completely overridden by HRGβ1 in all four cell lines. Conclusions These findings suggest that although antihormones such as fulvestrant may have potent acute growth-inhibitory activity in ER-positive breast cancer cells, their ability to induce and sensitise cells to growth factors may serve to reduce and ultimately limit their inhibitory activity

Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation

Producción CientíficaBackground: Given their unique capacity for antigen uptake, processing, and presentation, antigen-presenting cells (APCs) are critical for initiating and regulating innate and adaptive immune responses. We have previously shown the role of nicotinamide adenine dinucleotide (NAD+) in T-cell differentiation independently of the cytokine milieu, whereas the precise mechanisms remained unknown. Objective: The objective of this study is to further dissect the mechanism of actions of NAD+ and determine the effect of APCs on NAD+-mediated T-cell activation. Methods: Isolated dendritic cells and bone marrow–derived mast cells (MCs) were used to characterize the mechanisms of action of NAD+ on CD4+ T-cell fate in vitro. Furthermore, NAD+-mediated CD4+ T-cell differentiation was investigated in vivo by using wild-type C57BL/6, MC−/−, MHC class II−/−, Wiskott-Aldrich syndrome protein (WASP)−/−, 5C.C7 recombination-activating gene 2 (Rag2)−/−, and CD11b-DTR transgenic mice. Finally, we tested the physiologic effect of NAD+ on the systemic immune response in the context of Listeria monocytogenes infection. Results: Our in vivo and in vitro findings indicate that after NAD+ administration, MCs exclusively promote CD4+ T-cell differentiation, both in the absence of antigen and independently of major APCs. Moreover, we found that MCs mediated CD4+ T-cell differentiation independently of MHC II and T-cell receptor signaling machinery. More importantly, although treatment with NAD+ resulted in decreased MHC II expression on CD11c+ cells, MC-mediated CD4+ T-cell differentiation rendered mice resistant to administration of lethal doses of L monocytogenes. Conclusions: Collectively, our study unravels a novel cellular and molecular pathway that regulates innate and adaptive immunity through MCs exclusively and underscores the therapeutic potential of NAD+ in the context of primary immunodeficiencies and antimicrobial resistance.National Institutes of Health (grants R01NS073635 , R01MH110438 , R01HL096795 , U01HL126497 and R01AG039449)Instituto de Salud Carlos III (grant PI10/02 511)Fundación Ramón Areces (grant CIVP16A1843

PAR1 is selectively over expressed in high grade breast cancer patients: a cohort study

<p>Abstract</p> <p>Background</p> <p>The protease-activated receptor (PAR1) expression is correlated with the degree of invasiveness in cell lines. Nevertheless it has never been directed involved in breast cancer patients progression. The aim of this study was to determine whether PAR1 expression could be used as predictor of metastases and mortality.</p> <p>Methods</p> <p>In a cohort of patients with infiltrating ductal carcinoma studied longitudinally since 1996 and until 2007, PAR1 over-expression was assessed by immunoblotting, immunohistochemistry, and flow citometry. Chi-square and log rank tests were used to determine whether there was a statistical association between PAR1 overexpression and metastases, mortality, and survival. Multivariate analysis was performed including HER1, stage, ER and nodes status to evaluate PAR1 as an independent prognostic factor.</p> <p>Results</p> <p>Follow up was 95 months (range: 2–130 months). We assayed PAR1 in a cohort of patients composed of 136 patients; we found PAR1 expression assayed by immunoblotting was selectively associated with high grade patients (50 cases of the study cohort; P = 0.001). Twenty-nine of 50 (58%) patients overexpressed PAR1, and 23 of these (46%) developed metastases. HER1, stage, ER and PAR1 overexpression were robustly correlated (Cox regression, P = 0.002, P = 0.024 and P = 0.002 respectively). Twenty-one of the 50 patients (42%) expressed both receptors (PAR1 and HER1 P = 0.0004). We also found a statistically significant correlation between PAR1 overexpression and increased mortality (P = 0.0001) and development of metastases (P = 0.0009).</p> <p>Conclusion</p> <p>Our data suggest PAR1 overexpression may be involved in the development of metastases in breast cancer patient and is associated with undifferentiated cellular progression of the tumor. Further studies are needed to understand PAR1 mechanism of action and in a near future assay its potential use as risk factor for metastasis development in high grade breast cancer patients.</p

Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours

BACKGROUND: The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)α (cPLA(2)α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)α expression correlated with EGFR/HER2 over-expression in a small number of breast cancer cell lines. METHODS: The importance of differential cPLA(2)α activity in clinical breast cancer was established by relating the expression of cPLA(2)α in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters. RESULTS: High cPLA(2)α mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA(2)α expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA(2)α expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up. CONCLUSION: This study shows a role of cPLA(2)α in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker

Estrogen Receptor Silencing Induces Epithelial to Mesenchymal Transition in Human Breast Cancer Cells

We propose the hypothesis that loss of estrogen receptor function which leads to endocrine resistance in breast cancer, also results in trans-differentiation from an epithelial to a mesenchymal phenotype that is responsible for increased aggressiveness and metastatic propensity. siRNA mediated silencing of the estrogen receptor in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII) with altered morphology, increased motility with rearrangement and switch from a keratin/actin to a vimentin based cytoskeleton, and ability to invade simulated components of the extracellular matrix. Phenotypic profiling using an Affymetrix Human Genome U133 plus 2.0 GeneChip indicated geometric fold changes ≥3 in approximately 2500 identifiable unique sequences, with about 1270 of these being up-regulated in pII cells. Changes were associated with genes whose products are involved in cell motility, loss of cellular adhesion and interaction with the extracellular matrix. Selective analysis of the data also showed a shift from luminal to basal cell markers and increased expression of a wide spectrum of genes normally associated with mesenchymal characteristics, with consequent loss of epithelial specific markers. Over-expression of several peptide growth factors and their receptors are indicative of an increased contribution to the higher proliferative rates of pII cells as well as aiding their potential for metastatic activity. Signalling molecules that have been identified as key transcriptional drivers of epithelial to mesenchymal transition were also found to be elevated in pII cells. These data support our hypothesis that induced loss of estrogen receptor in previously estrogen/antiestrogen sensitive cells is a trigger for the concomitant loss of endocrine dependence and onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type. Inhibition of this transition through targeting of specific mediators may offer a useful supplementary strategy to circumvent the effects of loss of endocrine sensitivity