NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest.

Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment

Notch3 contributes to T-cell leukemia growth via regulation of the unfolded protein response

Unfolded protein response (UPR) is a conserved adaptive response that tries to restore protein homeostasis after endoplasmic reticulum (ER) stress. Recent studies highlighted the role of UPR in acute leukemias and UPR targeting has been suggested as a therapeutic approach. Aberrant Notch signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), as downregulation of Notch activity negatively affects T-ALL cell survival, leading to the employment of Notch inhibitors in T-ALL therapy. Here we demonstrate that Notch3 is able to sustain UPR in T-ALL cells, as Notch3 silencing favored a Bip-dependent IRE1α inactivation under ER stress conditions, leading to increased apoptosis via upregulation of the ER stress cell death mediator CHOP. By using Juglone, a naturally occurring naphthoquinone acting as an anticancer agent, to decrease Notch3 expression and induce ER stress, we observed an increased ER stress-associated apoptosis. Altogether our results suggest that Notch3 inhibition may prevent leukemia cells from engaging a functional UPR needed to compensate the Juglone-mediated ER proteotoxic stress. Notably, in vivo administration of Juglone to human T-ALL xenotransplant models significantly reduced tumor growth, finally fostering the exploitation of Juglone-dependent Notch3 inhibition to perturb the ER stress/UPR signaling in Notch3-dependent T-ALL subsets

Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

Summary: The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers) has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity. : Munk et al. use mass spectrometry-based proteomics to analyze the interplay between SUMOylation and phosphorylation in replication stress. They analyze changes in the SUMO and phosphoproteome after MMC and hydroxyurea treatments and find that the DNA damage response kinases ATR and ATM globally regulate SUMOylation upon replication stress and fork breakage. Keywords: Replication stress, quantitative proteomics, phosphoproteomics, SUMO, ATR, ATM, TOPBP1, MMC, kinase inhibitors, hydroxyure

Effects of Dietary Supplementation of Lactobacillus acidophilus on Blood Parameters and Gut Health of Rabbits

SIMPLE SUMMARY: Gastrointestinal diseases are one of the most common causes of death in rabbits. Thus, maintaining a proper gut health is fundamental to guarantee adequate growth performance and welfare of the animals. Probiotics (e.g., Lactobacillus acidophilus) have been proposed as valuable alternatives to positively modulate gut health. The aim of this study was to evaluate the effects of Lactobacillus acidophilus D2/CSL on biochemical parameters, faecal score, cecal pH, gut histomorphometry, microbiota composition and faecal short-chain fatty acids in rabbits. Overall, the dietary inclusion of 1 × 10(9) cfu/kg feed once a day of Lactobacillus acidophilus D2/CSL did not impair rabbit productive performance, blood biochemical parameters, faecal score, gut morphometry, cecal pH, microbiota and short-chain fatty acids concentration. However, it reduced disease incidence and animal death, suggesting that it could improve disease resistance in rabbits. ABSTRACT: This study aimed to evaluate the effects of Lactobacillus acidophilus D2/CSL (L-1 × 10(9) cfu/kg feed/day) on biochemical parameters, faecal score (FS), cecal pH, gut morphometry, microbiota and cecal short-chain fatty acid (SCFAs) in rabbits. Three zootechnical trials were performed and in each trial 30 rabbits were allotted to two groups; a probiotic group (L) and a control group (C). At slaughter (day 45), samples of blood, duodenum, jejunum, ileum, liver and spleen were collected and submitted to histomorphometric analyses. Blood biochemical analyses, cecal microbiota and SCFAs determination were also performed. In trial 1 and 3, L. acidophilus D2/CSL did not affect productive parameters (p > 0.05). However, L group of trial 1 showed a lower morbidity and mortality compared to the control. In trial 2, C group showed a higher daily feed intake (p = 0.018) and a positive statistical tendency for live weight and average daily gain (p = 0.068). On the contrary, albumin was higher and ALFA-1 globulin was lower in the C group compared to L (p < 0.05). In all the trials, FS, cecal pH, histomorphometry, microbiota and SCFAs were unaffected. In conclusion, L. acidophilus D2/CSL did not impair growth performances, gut and rabbit’s health, reducing morbidity and mortality

Proteomics of resistance to Notch1 inhibition in acute lymphoblastic leukemia reveals targetable kinase signatures

Contains fulltext : 234212.pdf (Publisher’s version ) (Open Access

NOTCH3 inactivation increases Triple Negative Breast Cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest.

Triple-negative breast cancer (TNBC) accounts for about 15-20% of breast cancers and represents the most aggressive subtype (1). To date, no molecularly targeted agents are approved for TNBC, leading to the conventional chemotherapy the role of primary option for systemic treatment. Therefore, effective therapeutic strategies for TNBC are urgently needed. The tyrosine kinase receptor EGFR overexpression is a hallmark of TNBC. Anti-EGFR therapies, including EGFR inhibitors, are not currently approved for breast cancer treatment, since the results from clinical trials are disappointing (2) due to the existence of compensatory pathways that confer resistance to EGFR inhibition. Notch signaling dysregulation is often associated with the pathogenesis and progression of TNBC (3). In addition, Notch-EGFR interplay occurs in breast cancer (4), raising the possibility that Notch signalling could be involved in the resistance to EGFR inhibition. Consequently, the combined Notch-EGFR pathway inhibition, in this context, is a potential therapeutic approach for overcoming resistance to drugs (5). Pan-Notch inhibition using gamma-secretase-inhibitors (GSIs) treatment supports this conclusion but it fails to distinguish the particular Notch receptor which drives growth. Therefore, it is relevant to investigate which is the main Notch receptor involved in the resistance to EGFR inhibition in order to understand the main strategy for TNBC cancer therapy. It has been demonstrated that constitutive Notch3 signalling can drive an oncogenic program in a subset of TNBCs, thus suggesting that Notch3 activity, and not others Notch paralogues, may be relevant in this breast cancer subtype (6)