Nicastrin as a novel therapeutic target in breast cancer

Nicastrin is an essential component of the gamma-secretase (GS) enzyme complex, required for its synthesis and recognition of substrates for proteolytic cleavage. The purpose of this study was to investigate whether nicastrin has a prognostic value or the potential as a therapeutic target in breast cancer. Tissue microarrays (TMAs) (n = 1050), and breast cancer cell line analyses confirmed that nicastrin expression was upregulated in breast cancer compared to normal breast cells. In TMA patient samples, high nicastrin expression was observed in 47.5% of cases and correlated with worse breast cancer specific survival in the ERα negative cohort. Transient and stable gene silencing of nicastrin in vitro, resulted in the disruption of the GS complex activity and a decrease in Notch1 cleavage. Nicastrin silencing in invasive MDA-MB-231 and HCC1806 cells resulted in the loss of vimentin expression and a reduction in cell invasion, which was concomitant with the formation of cell-cell junctions, as well as cellular repolarisation. In a population of breast cancer cells harbouring the cancer stem cell phenotype (CD44+/CD24-), nicastrin depletion abrogated expression of the epithelial to mesenchymal (EMT) markers, vimentin, SIP1 and Snail. Furthermore, nicastrin overexpression in the non-malignant MCF10A breast cells increased expression of other GS components, Notch activation, vimentin expression and invasive capacity. These data indicate that nicastrin can function to maintain the EMT transition during breast cancer progression. We have developed anti-nicastrin polyclonal and monoclonal antibodies and have shown that they are able to decrease the invasive and proliferative capacity of MDA-MB-231 breast cancer cells in vitro. In order to dissect the role of nicastrin within the GS complex from its presumed GS-independent signaling role in breast cancer cells, we have performed gene array analyses and identified a subset of genes that are nicastrin-dependent and are not affected by silencing of the Notch receptors. The data presented in this thesis therefore support our hypothesis that an antagonising monoclonal antibody could be suitable to inhibit nicastrin as a potential therapeutic target in invasive breast cancer

Targeting the superoxide/nitric oxide ratio by L-arginine and SOD mimic in diabetic rat skin

AbstractSetting the correct ratio of superoxide anion (O2•-) and nitric oxide (•NO) radicals seems to be crucial in restoring disrupted redox signaling in diabetic skin and improvement of •NO physiological action for prevention and treatment of skin injuries in diabetes. In this study we examined the effects of L-arginine and manganese(II)-pentaazamacrocyclic superoxide dismutase (SOD) mimic – M40403 in diabetic rat skin. Following induction of diabetes by alloxan (blood glucose level ≥12 mMol l −1) non-diabetic and diabetic male Mill Hill hybrid hooded rats were divided into three subgroups: (i) control, and receiving: (ii) L-arginine, (iii) M40403. Treatment of diabetic animals started after diabetes induction and lasted for 7 days. Compared to control, lower cutaneous immuno-expression of endothelial NO synthase (eNOS), heme oxygenase 1 (HO1), manganese SOD (MnSOD) and glutathione peroxidase (GSH-Px), in parallel with increased NFE2-related factor 2 (Nrf2) and nitrotyrosine levels characterized diabeti..

Land Management Impacts on Soil Properties and Initial Soil Erosion Processes in Olives and Vegetable Crops

This research aims to assess the impacts of soil use management on runoff, soil losses, and their main soil controls in vegetable cropland (CROP), tilled olives (OT), and grass-covered olive orchards (OGC) on Leptosol in Croatia. Soil analysis and rainfall simulation experiments were conducted to quantify runoff (Run), soil, and nutrient losses. Bulk density (BD) was significantly higher at OT plots, in addition to the CROP plots. Water-stable aggregates (WSA), mean weight diameter (MWD), and soil organic matter (OM) were significantly higher in OGC plots compared to the other land uses. Run and soil loss (SL) were significantly higher in CROP and OT plots compared to the OGC plots. The CROP plots showed soil management that can be considered as unsustainable with 52, 68- and 146-times higher losses of phosphorus (P loss), nitrogen (N loss), and carbon (C loss) compared to the OGC plots. The principal component analysis showed that MWD was associated with vegetation cover (VC), water-holding capacity (WHC), WSA, OM, total nitrogen (TN), time to ponding (TP), and time to runoff (TR). These variables were negatively related to P2O5, Run, SL, and P, N, and C loss. Results indicate the need for the adoption of conservation strategies in croplands and olive orchards

Gait Analysis using Wearable Sensors with Multiple Sclerosis Patients

In this study we investigated gait measurement with wearable sensor for subjects with and without multiple sclerosis (MS) and evaluation gait function.The gait function was measured with Avatar sensors system in 3 patients with MS and in 3 healthy subjects without MS. The system consists of a main sensor node and three additional fixtures. Each sensor node is wearing three-axial accelerometer and two-axis gyroscope. Cross-correlation analysis with the walk signal was applied.Coefficient values from cross-correlation are determined for all 6 subjects. Then for a new unknown subject the cross-correlation was applied and the mean value cross-correlation for healthy subjects was 0.0477, while in MS subjects this value was 0.0207. A proven validation for this small training system has shown the evidence for different gait analysis for MS and healthy subjects.This small study opens a new avenue for clinical diagnosis of potential MS subjects while wearable sensor can provide an objective framework for assessing gait abnormality. The measured data can provide better understanding on the progression of the disease and response to treatment

Gender Related Differences in the Clinical Presentation of Hypertrophic Cardiomyopathy-An Analysis from the SILICOFCM Database

Background and Objectives: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease that affects approximately 1 in 500 people. Due to an incomplete disease penetrance associated with numerous factors, HCM is not manifested in all carriers of genetic mutation. Although about two-thirds of patients are male, it seems that female gender is associated with more severe disease phenotype and worse prognosis. The objective of this study was to evaluate the gender related differences in HCM presentation. Materials and Methods: This study was conducted as a part of the international multidisciplinary SILICOFCM project. Clinical information, laboratory analyses, electrocardiography, echocardiography, and genetic testing data were collected for 362 HCM patients from four clinical centers (Florence, Newcastle, Novi Sad, and Regensburg). There were 33% female patients, and 67% male patients. Results: Female patients were older than males (64.5 vs. 53.5 years, p < 0.0005). The male predominance was present across all age groups until the age of 70, when gender distribution became comparable. Females had higher number of symptomatic individuals then males (69% vs. 52%, p = 0.003), most frequently complaining of dyspnea (50% vs. 30%), followed by chest pain (30% vs. 17%), fatigue (26% vs. 13%), palpitations (22% vs. 13%), and syncope (13% vs. 8%). The most common rhythm disorder was atrial fibrillation which was present in a similar number of females and males (19% vs. 13%, p = 0.218). Levels of N-terminal pro-brain natriuretic peptide were comparable between the genders (571 vs. 794 ng/L, p = 0.244). Echocardiography showed similar thickness of interventricular septum (18 vs. 16 mm, p = 0.121) and posterolateral wall (13 vs. 12 mm, p = 0.656), however, females had a lower number of systolic anterior motion (8% vs. 16%, p = 0.020) and other mitral valve abnormalities. Conclusions: Female patients are underrepresented but seem to have a more pronounced clinical presentation of HCM. Therefore, establishing gender specific diagnostic criteria for HCM should be considered

Genetic determinants of clinical phenotype in hypertrophic cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease that affects approximately one in 500 people. HCM is a recognized genetic disorder most often caused by mutations involving myosin-binding protein C (MYBPC3) and beta -myosin heavy chain (MYH7) which are responsible for approximately three-quarters of the identified mutations.MethodsAs a part of the international multidisciplinary SILICOFCM project (www.silicofcm.eu) the present study evaluated the association between underlying genetic mutations and clinical phenotype in patients with HCM. Only patients with confirmed single pathogenic mutations in either MYBPC3 or MYH7 genes were included in the study and divided into two groups accordingly. The MYBPC3 group was comprised of 48 patients (76%), while the MYH7 group included 15 patients (24%). Each patient underwent clinical examination and echocardiography.ResultsThe most prevalent symptom in patients with MYBPC3 was dyspnea (44%), whereas in patients with MYH7 it was palpitations (33%). The MYBPC3 group had a significantly higher number of patients with a positive family history of HCM (46% vs. 7%; p=0.014). There was a numerically higher prevalence of atrial fibrillation in the MYH7 group (60% vs. 35%, p=0.085). Laboratory analyses revealed normal levels of creatinine (85.518.3 vs. 81.3 +/- 16.4 mu mol/l; p=0.487) and blood urea nitrogen (10.2 +/- 15.6 vs. 6.9 +/- 3.9 mmol/l; p=0.472) which were similar in both groups. The systolic anterior motion presence was significantly more frequent in patients carrying MYH7 mutation (33% vs. 10%; p=0.025), as well as mitral leaflet abnormalities (40% vs. 19%; p=0.039). Calcifications of mitral annulus were registered only in MYH7 patients (20% vs. 0%; p=0.001). The difference in diastolic function, i.e. E/e ' ratio between the two groups was also noted (MYBPC3 8.8 +/- 3.3, MYH7 13.9 +/- 6.9, p=0.079).Conclusions Major findings of the present study corroborate the notion that MYH7 gene mutation patients are presented with more pronounced disease severity than those with MYBPC3

Notch-1-PTEN-ERK1/2 signaling axis promotes HER2+ breast cancer cell proliferation and stem cell survival

Trastuzumab targets the HER2 receptor on breast cancer cells to attenuate HER2-driven tumor growth. However, resistance to trastuzumab-based therapy remains a major clinical problem for women with HER2+ breast cancer. Breast cancer stem cells (BCSCs) are suggested to be responsible for drug resistance and tumor recurrence. Notch signaling has been shown to promote BCSC survival and self-renewal. Trastuzumab-resistant cells have increased Notch-1 expression. Notch signaling drives cell proliferation in vitro and is required for tumor recurrence in vivo. We demonstrate herein a mechanism by which Notch-1 is required for trastuzumab resistance by repressing PTEN expression to contribute to activation of ERK1/2 signaling. Furthermore, Notch-1-mediated inhibition of PTEN is necessary for BCSC survival in vitro and in vivo. Inhibition of MEK1/2-ERK1/2 signaling in trastuzumab-resistant breast cancer cells mimics effects of Notch-1 knockdown on bulk cell proliferation and BCSC survival. These findings suggest that Notch-1 contributes to trastuzumab resistance by repressing PTEN and this may lead to hyperactivation of ERK1/2 signaling. Furthermore, high Notch-1 and low PTEN mRNA expression may predict poorer overall survival in women with breast cancer. Notch-1 protein expression predicts poorer survival in women with HER2+ breast cancer. These results support a potential future clinical trial combining anti-Notch-1 and anti-MEK/ERK therapy for trastuzumab-resistant breast cancer

AKT and 14-3-3 regulate Notch4 nuclear localization

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling

Inhibition of HER2 enriches for Jagged1-dependent breast cancer stem cells: role for membrane Jagged1

Purpose: Human Epidermal Growth Factor Receptor 2 (HER2) positive breast cancer is driven by cells possessing stem-like properties of self-renewal and differentiation, referred to as Cancer Stem Cells (CSCs). CSCs are implicated in radiotherapy, chemotherapy resistance, and tumor recurrence. Notch promotes breast CSCs survival and self-renewal, and overexpression of Notch1 and the Notch ligand Jagged1 predict poor outcome. Resistance to anti-HER2 therapy in HER2+ breast cancer requires Notch1, and that combination of trastuzumab and a Gamma Secretase Inhibitor (GSI) prevents tumor relapse in xenograft models. Experimental Design: The current study investigates mechanisms by which HER2 tyrosine kinase activity regulates Notch-dependent CSC survival and tumor initiation. Results: Lapatinib-mediated HER2 inhibition shifts the population of HER2+ breast cancer cells from low membrane Jagged1 expressing to higher levels, independent of sensitivity to anti-HER2 treatment within the bulk cell population. This increase in membrane Jagged1 is associated with higher Notch receptor expression, activation, and enrichment of CSCs in vitro and in vivo. Importantly, lapatinib treatment results in growth arrest and cell death of Jagged1 low-expressing cells while the Jagged1 high-expressing cells continue to cycle. High membrane Jagged1 protein expression predicts poor overall cumulative survival in women with HER2+ breast cancer. Conclusions: These results indicate that higher membrane Jagged1 expression may be used to either predict response to anti-HER2 therapy or for detection of Notch sensitive CSCs post therapy. Sequential blockade of HER2 followed by Jagged1 or Notch could be more effective than simultaneous blockade to prevent drug resistance and tumor progression