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

Opsonin-deficient nucleoproteic corona endows unPEGylated liposomes with stealth properties in vivo

For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, the limited clinical translation of PEGylated liposomes is mainly due to the protein corona formation and the subsequent modification of liposomes’ synthetic identity, which affects their interactions with immune cells and blood residency. Here we exploit the electric charge of DNA to generate unPEGylated liposome/DNA complexes that, upon exposure to human plasma, gets covered with an opsonin-deficient protein corona. The final product of the synthetic process is a biomimetic nanoparticle type covered by a proteonucleotidic corona, or “proteoDNAsome”, which maintains its synthetic identity in vivo and is able to slip past the immune system more efficiently than PEGylated liposomes. Accumulation of proteoDNAsomes in the spleen and the liver was lower than that of PEGylated systems. Our work highlights the importance of generating stable biomolecular coronas in the development of stealth unPEGylated particles, thus providing a connection between the biological behavior of particles in vivo and their synthetic identity

Getting insights into the multi-faceted role of Notch3 in different tumor contexts

Notch receptor family comprises evolutionary conserved single-pass transmembrane proteins which are involved in several cellular processes during embryogenesis and in adult tissues. Given their pleiotropic effects, their deregulation is associated to the development of several diseases including cancer. Since Notch receptors are differentially implicated in essentially all of the hallmarks of cancer, dissecting every facets of each receptor in different tumor contexts could help to foster effective and specific targeted therapies. In keeping with this consideration, during my PhD project I specifically focused my attention on Notch3 receptor in three different tumor contexts: Triple-Negative Breast Cancer (Project n°1), T-cell Acute Lymphoblastic Leukemia (Project n°2), and Ovarian Cancer (Project n°3). The main objective of this work, performed in the laboratory of Prof. Isabella Screpanti (at the Department of Molecular Medicine of Sapienza University) under the direct supervision of Dr. Saula Checquolo, was to broaden the knowledge of Notch3 receptor, trying to puzzle out its role in cancer, mainly focusing on how it is specifically regulated at the post-translational level, which still represents an unknown field of Notch3 regulation process. Indeed, Project n°1 and Project n°2 report two different crosstalk between Notch3 and other signaling pathways, the EGFR signaling and the Unfolded Protein Response (UPR), respectively. Specifically, on the one hand, we demonstrate that Notch3 regulates EGFR localization in Triple-Negative Breast Cancer, making the receptor unavailable to be targeted by the anti-EGFR agents, such as tyrosine kinase inhibitors, thus highlighting how Notch3 could be crucial in promoting drug-resistance. On the other hand, we document that Notch3 is involved in the activation of pro-survival UPR by directly interacting with an UPR “effector”, thus sustaining cancer cell growth under ER stress conditions. Interestingly, in both tumor contexts Notch3 fulfils its function in a transcriptionindependent manner, paving the way for the study of a mostly untouched aspect of Notch3 function in cancer. Moreover, in Project n°3 we cover a largely unstudied but crucial layer of finetuning and regulation of Notch3: its post-translational modifications (PTMs). To date, little is known about the potential different PTMs of Notch3, mainly regarding the glycosylation of its extracellular domain and the acetylation of its intracellular domain. Here we focus our attention on the study of the phosphorylation status of Notch3 intracellular domain showing how it influences its longevity and who are the actors of this regulation, thus finally fostering a novel therapeutic approach to target Notch3-dependent tumors through the modulations of these specific Notch3 protein regulators. All in all, there are still lots of gaps in the puzzle of “Notch3 world” but some small pieces were falling neatly into place

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)

5FU/Oxaliplatin-induced Jagged1 cleavage counteracts apoptosis induction in colorectal cancer: a novel mechanism of intrinsic drug resistance

Colorectal cancer (CRC) is characterized by early metastasis, resistance to anti-cancer therapy, and high mortality rate. Despite considerable progress in the development of new treatment options that improved survival benefits in patients with early-stage or advanced CRC, many patients relapse due to the activation of intrinsic or acquired chemoresistance mechanisms. Recently, we reported novel findings about the role of Jagged1 in CRC tumors with Kras signatures. We showed that Jagged1 is a novel proteolytic target of Kras signaling, which induces Jagged1 processing/activation resulting in Jag1-ICD release, which favors tumor development in vivo, through a non-canonical mechanism. Herein, we demonstrate that OXP and 5FU cause a strong accumulation of Jag1-ICD oncogene, through ERK1/2 activation, unveiling a surviving subpopulation with an enforced Jag1-ICD expression, presenting the ability to counteract OXP/5FU-induced apoptosis. Remarkably, we also clarify the clinical ineffectiveness of gamma-secretase inhibitors (GSIs) in metastatic CRC (mCRC) patients. Indeed, we show that GSI compounds trigger Jag1-ICD release, which promotes cellular growth and EMT processes, functioning as tumor-promoting agents in CRC cells overexpressing Jagged1. We finally demonstrate that Jagged1 silencing in OXP- or 5FU-resistant subpopulations is enough to restore the sensitivity to chemotherapy, confirming that drug sensitivity/resistance is Jag1-ICD-dependent, suggesting Jagged1 as a molecular predictive marker for the outcome of chemotherapy