Loss of ATP2C1 function promotes trafficking and degradation of NOTCH1: Implications for Hailey-Hailey disease.

Hailey-Hailey disease (HHD) is a rare autosomal dominantly inherited disorder caused by mutations in the ATP2C1 gene that encodes an adenosine triphosphate (ATP)-powered calcium channel pump. HHD is characterized by impaired epidermal cell-to-cell adhesion and defective keratinocyte growth/differentiation. The mechanism by which mutant ATP2C1 causes HHD is unknown and current treatments for affected individuals do not address the underlying defects and are ineffective. Notch signalling is a direct determinant of keratinocyte growth and differentiation. We found that loss of ATP2C1 leads to impaired Notch1 signalling, thus deregulation of the Notch signalling response is therefore likely to contribute to HHD manifestation. NOTCH1 is a transmembrane receptor and upon ligand binding, the intracellular domain (NICD) translocates to the nucleus activating its target genes. In the context of HHD, we found that loss of ATP2C1 function promotes upregulation of the active NOTCH1 protein (NICD-Val1744). Here, deeply exploring this aspect, we observed that NOTCH1 activation is not associated with the transcriptional enhancement of its targets. Moreover, in agreement with these results, we found a cytoplasmic localization of NICD-Val1744. We have also observed that ATP2C1-loss is associated with the degradation of NICD-Val1744 through the lysosomal/proteasome pathway. These results show that ATP2C1-loss could promote a mechanism by which NOTCH1 is endocytosed and degraded by the cell membrane. The deregulation of this phenomenon, finely regulated in physiological conditions, could in HHD lead to the deregulation of NOTCH1 with alteration of skin homeostasis and disease manifestation.

Identification of a Novel Curcumin Derivative Influencing Notch Pathway and DNA Damage as a Potential Therapeutic Agent in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy considered curable by modern clinical management. Nevertheless, the prognosis for T-ALL high-risk cases or patients with relapsed and refractory disease is still dismal. Therefore, there is a keen interest in developing more efficient and less toxic therapeutic approaches. T-ALL pathogenesis is associated with Notch signaling alterations, making this pathway a highly promising target in the fight against T-ALL. Here, by exploring the anti-leukemic capacity of the natural polyphenol curcumin and its derivatives, we found that curcumin exposure impacts T-ALL cell line viability and decreases Notch signaling in a dose- and time-dependent fashion. However, our findings indicated that curcumin-mediated cell outcomes did not depend exclusively on Notch signaling inhibition, but might be mainly related to compound-induced DNA-damage-associated cell death. Furthermore, we identified a novel curcumin-based compound named CD2066, endowed with potentiated anti-proliferative activity in T-ALL compared to the parent molecule curcumin. At nanomolar concentrations, CD2066 antagonized Notch signaling, favored DNA damage, and acted synergistically with the CDK1 inhibitor Ro3306 in T-ALL cells, thus representing a promising novel candidate for developing therapeutic agents against Notch-dependent T-ALL.

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 γ-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.

Nobiletin and Xanthohumol Sensitize Colorectal Cancer Stem Cells to Standard Chemotherapy.

Colorectal cancer (CRC) mortality is mainly caused by patient refractoriness to common anti-cancer therapies and consequent metastasis formation. Besides, the notorious toxic side effects of chemotherapy are a concurrent obstacle to be tackled. Thus, new treatment approaches are needed to effectively improve patient outcomes. Compelling evidence demonstrated that cancer stem cells (CSCs) are responsible for treatment failure and relapse. New natural treatment approaches showed capabilities to selectively target the CSC subpopulation by rendering them targetable by standard cytotoxic compounds. Herein we show the anti-cancer properties of the polymethoxyflavones and prenylflavonoids extracted from Citrus sinensis and Humulus lupulus, respectively. The natural biofunctional fractions, singularly and in combination, reduced the cell viability of CRC stem cells (CR-CSCs) and synergized with 5-fluorouracil and oxaliplatin (FOX) chemotherapy. These phenomena were accompanied by a reduced S and G2/M phase of the cell cycle and upregulation of cell death-related genes. Notably, both phytoextracts in combination with FOX thwarted stemness features in CR-CSCs as demonstrated by the impaired clonogenic potential and decreased Wnt pathway activation. Extracts lowered the expression of CD44v6 and affected the expansion of metastatic CR-CSCs in patients refractory to chemotherapy. Together, this study highlights the importance of polymethoxyflavones and prenylflavonoids as natural remedies to aid oncological therapies.

When Viruses Cross Developmental Pathways.

Aberrant regulation of developmental pathways plays a key role in tumorigenesis. Tumor cells differ from normal cells in their sustained proliferation, replicative immortality, resistance to cell death and growth inhibition, angiogenesis, and metastatic behavior. Often they acquire these features as a consequence of dysregulated Hedgehog, Notch, or WNT signaling pathways. Human tumor viruses affect the cancer cell hallmarks by encoding oncogenic proteins, and/or by modifying the microenvironment, as well as by conveying genomic instability to accelerate cancer development. In addition, viral immune evasion mechanisms may compromise developmental pathways to accelerate tumor growth. Viruses achieve this by influencing both coding and non-coding gene regulatory pathways. Elucidating how oncogenic viruses intersect with and modulate developmental pathways is crucial to understanding viral tumorigenesis. Many currently available antiviral therapies target viral lytic cycle replication but with low efficacy and severe side effects. A greater understanding of the cross-signaling between oncogenic viruses and developmental pathways will improve the efficacy of next-generation inhibitors and pave the way to more targeted antiviral therapies.

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.

A Dynamic Role of Mastermind-Like 1: A Journey Through the Main (Path)ways Between Development and Cancer.

Major signaling pathways, such as Notch, Hedgehog (Hh), Wnt/ß-catenin and Hippo, are targeted by a plethora of physiological and pathological stimuli, ultimately resulting in the modulation of genes that act coordinately to establish specific biological processes. Many biological programs are strictly controlled by the assembly of multiprotein complexes into the nucleus, where a regulated recruitment of specific transcription factors and coactivators on gene promoter region leads to different transcriptional outcomes. MAML1 results to be a versatile coactivator, able to set up synergistic interlinking with pivotal signaling cascades and able to coordinate the network of cross-talking pathways. Accordingly, despite its original identification as a component of the Notch signaling pathway, several recent reports suggest a more articulated role for MAML1 protein, showing that it is able to sustain/empower Wnt/ß-catenin, Hh and Hippo pathways, in a Notch-independent manner. For this reason, MAML1 may be associated to a molecular "switch", with the function to control the activation of major signaling pathways, triggering in this way critical biological processes during embryonic and post-natal life. In this review, we summarize the current knowledge about the pleiotropic role played by MAML proteins, in particular MAML1, and we recapitulate how it takes part actively in physiological and pathological signaling networks. On this point, we also discuss the contribution of MAML proteins to malignant transformation. Accordingly, genetic alterations or impaired expression of MAML proteins may lead to a deregulated crosstalk among the pathways, culminating in a series of pathological disorders, including cancer development. Given their central role, a better knowledge of the molecular mechanisms that regulate the interplay of MAML proteins with several signaling pathways involved in tumorigenesis may open up novel opportunities for an attractive molecular targeted anticancer therapy.

PLK1 targets NOTCH1 during DNA damage and mitotic progression.

Notch signaling plays a complex role in carcinogenesis, and its signaling pathway has both tumor suppressor and oncogenic components. To identify regulators that might control this dual activity of NOTCH1, we screened a chemical library targeting kinases and identified Polo-like kinase 1 (PLK1) as one of the kinases involved in arsenite-induced NOTCH1 down-modulation. As PLK1 activity drives mitotic entry but also is inhibited after DNA damage, we investigated the PLK1-NOTCH1 interplay in the G2 phase of the cell cycle and in response to DNA damage. Here, we found that PLK1 regulates NOTCH1 expression at G2/M transition. However, when cells in G2 phase are challenged with DNA damage, PLK1 is inhibited to prevent entry into mitosis. Interestingly, we found that the interaction between NOTCH1 and PLK1 is functionally important during the DNA damage response, as we found that whereas PLK1 activity is inhibited, NOTCH1 expression is maintained during DNA damage response. During genotoxic stress, cellular transformation requires that promitotic activity must override DNA damage checkpoint signaling to drive proliferation. Interestingly, we found that arsenite-induced genotoxic stress causes a PLK1-dependent signaling response that antagonizes the involvement of NOTCH1 in the DNA damage checkpoint. Taken together, our data provide evidence that Notch signaling is altered but not abolished in SCC cells. Thus, it is also important to recognize that Notch plasticity might be modulated and could represent a key determinant to switch on/off either the oncogenic or tumor suppressor function of Notch signaling in a single type of tumor.

Wnt, Notch, and TGF-ß Pathways Impinge on Hedgehog Signaling Complexity: An Open Window on Cancer.

Constitutive activation of the Hedgehog (Hh) signaling pathway is associated with increased risk of developing several malignancies. The biological and pathogenic importance of Hh signaling emphasizes the need to control its action tightly, both physiologically and therapeutically. Evidence of crosstalk between Hh and other signaling pathways is reported in many tumor types. Here, we provide an overview of the current knowledge about the communication between Hh and major signaling pathways, such as Notch, Wnt, and transforming growth factor ß (TGF-ß), which play critical roles in both embryonic and adult life. When these pathways are unbalanced, impaired crosstalk contributes to disease development. It is reported that more than one of these pathways are active in different type of tumors, at the same time. Therefore, starting from a plethora of stimuli that activate multiple signaling pathways, we describe the signals that preferentially converge on the Hh signaling cascade that influence its activity. Moreover, we highlight several connection points between Hh and Notch, Wnt, or TGF-ß pathways, showing a reciprocal synergism that contributes to tumorigenesis, supporting a more malignant behavior by tumor cells, such as in leukemia and brain tumors. Understanding the importance of these molecular interlinking networks will provide a rational basis for combined anticancer drug development.

Kras/ADAM17-Dependent Jag1-ICD Reverse Signaling Sustains Colorectal Cancer Progression and Chemoresistance.

Colorectal cancer is characterized by well-known genetic defects and approximately 50% of cases harbor oncogenic Ras mutations. Increased expression of Notch ligand Jagged1 occurs in several human malignancies, including colorectal cancer, and correlates with cancer progression, poor prognosis, and recurrence. Herein, we demonstrated that Jagged1 was constitutively processed in colorectal cancer tumors with mutant Kras, which ultimately triggered intrinsic reverse signaling via its nuclear-targeted intracellular domain Jag1-ICD. This process occurred when Kras/Erk/ADAM17 signaling was switched on, demonstrating that Jagged1 is a novel target of the Kras signaling pathway. Notably, Jag1-ICD promoted tumor growth and epithelial-mesenchymal transition, enhancing colorectal cancer progression and chemoresistance both in vitro and in vivo. These data highlight a novel role for Jagged1 in colorectal cancer tumor biology that may go beyond its effect on canonical Notch activation and suggest that Jag1-ICD may behave as an oncogenic driver that is able to sustain tumor pathogenesis and to confer chemoresistance through a noncanonical mechanism. SIGNIFICANCE: These findings present a novel role of the transcriptionally active Jag1-ICD fragment to confer and mediate some of the activity of oncogenic KRAS.

Notch/CXCR4 Partnership in Acute Lymphoblastic Leukemia Progression.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children. Recent advances in chemotherapy have made ALL a curable hematological malignancy. In children, there is 25% chance of disease relapse, typically in the central nervous system. While in adults, there is a higher chance of relapse. ALL may affect B-cell or T-cell lineages. Different genetic alterations characterize the two ALL forms. Deregulated Notch, either Notch1 or Notch3, and CXCR4 receptor signaling are involved in ALL disease development and progression. By analyzing their relevant roles in the pathogenesis of the two ALL forms, new molecular mechanisms able to modulate cancer cell invasion may be visualized. Notably, the partnership between Notch and CXCR4 may have considerable implications in understanding the complexity of T- and B-ALL. These two receptor pathways intersect other critical signals in the proliferative, differentiation, and metabolic programs of lymphocyte transformation. Also, the identification of the crosstalks in leukemia-stroma interaction within the tumor microenvironment may unveil new targetable mechanisms in disease relapse. Further studies are required to identify new challenges and opportunities to develop more selective and safer therapeutic strategies in ALL progression, possibly contributing to improve conventional hematological cancer therapy.

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of ßTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated ßTrCP, the substrate-receptor subunit of the SCFßTrCP ubiquitin ligase, and promotes ßTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors.

Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer caused by the deregulation of key T-cell developmental pathways, including Notch signaling. Aberrant Notch signaling in T-ALL occurs by NOTCH1 gain-of-function mutations and by NOTCH3 overexpression. Although NOTCH3 is assumed as a Notch1 target, machinery driving its transcription in T-ALL is undefined in leukemia subsets lacking Notch1 activation. Here, we found that the binding of the intracellular Notch3 domain, as well as of the activated Notch1 fragment, to the NOTCH3 gene locus led to the recruitment of the H3K27 modifiers JMJD3 and p300, and it was required to preserve transcriptional permissive/active H3K27 marks and to sustain NOTCH3 gene expression levels. Consistently, pharmacological inhibition of JMJD3 by GSKJ4 treatment or of p300 by A-485 decreased the levels of expression of NOTCH3, NOTCH1 and of the Notch target genes DELTEX1 and c-Myc and abrogated cell viability in both Notch1- and Notch3-dependent T-cell contexts. Notably, re-introduction of exogenous Notch1, Notch3 as well as c-Myc partially rescued cells from anti-growth effects induced by either treatment. Overall our findings indicate JMJD3 and p300 as general Notch1 and Notch3 signaling co-activators in T-ALL and suggest further investigation on the potential therapeutic anti-leukemic efficacy of their enzymatic inhibition in Notch/c-Myc axis-related cancers and diseases.

Notch3 Targeting: A Novel Weapon against Ovarian Cancer Stem Cells.

Notch signaling is frequently activated in ovarian cancer (OC) and contributes to the proliferation and survival of cultured OC cells as well as to tumor formation and angiogenesis in xenograft models. Several studies demonstrate that Notch3 expression renders cancer cells more resistant to carboplatin, contributing to chemoresistance and poor survival of OC-bearing patients. This suggests that Notch3 can represent both a biomarker and a target for therapeutic interventions in OC patients. Although it is still unclear how chemoresistance arises, different lines of evidence support a critical role of cancer stem cells (CSCs), suggesting that CSC targeting by innovative therapeutic approaches might represent a promising tool to efficiently reduce OC recurrence. To date, CSC-directed therapies in OC tumors are mainly targeted to the inhibition of CSC-related signaling pathways, including Notch. As it is increasingly evident the involvement of Notch signaling, and in particular of Notch3, in regulating stem-like cell maintenance and expansion in several tumors, here we provide an overview of the current knowledge of Notch3 role in CSC-mediated OC chemoresistance, finally exploring the potential design of innovative Notch3 inhibition-based therapies for OC treatment, aimed at eradicating tumor through the suppression of CSCs.

A Simplified Genomic Profiling Approach Predicts Outcome in Metastatic Colorectal Cancer.

The response of metastatic colorectal cancer (mCRC) to the first-line conventional combination therapy is highly variable, reflecting the elevated heterogeneity of the disease. The genetic alterations underlying this heterogeneity have been thoroughly characterized through omic approaches requiring elevated efforts and costs. In order to translate the knowledge of CRC molecular heterogeneity into a practical clinical approach, we utilized a simplified Next Generation Sequencing (NGS) based platform to screen a cohort of 77 patients treated with first-line conventional therapy. Samples were sequenced using a panel of hotspots and targeted regions of 22 genes commonly involved in CRC. This revealed 51 patients carrying actionable gene mutations, 22 of which carried druggable alterations. These mutations were frequently associated with additional genetic alterations. To take into account this molecular complexity and assisted by an unbiased bioinformatic analysis, we defined three subgroups of patients carrying distinct molecular patterns. We demonstrated these three molecular subgroups are associated with a different response to first-line conventional combination therapies. The best outcome was achieved in patients exclusively carrying mutations on TP53 and/or RAS genes. By contrast, in patients carrying mutations in any of the other genes, alone or associated with mutations of TP53/RAS, the expected response is much worse compared to patients with exclusive TP53/RAS mutations. Additionally, our data indicate that the standard approach has limited efficacy in patients without any mutations in the genes included in the panel. In conclusion, we identified a reliable and easy-to-use approach for a simplified molecular-based stratification of mCRC patients that predicts the efficacy of the first-line conventional combination therapy.

EZH2, HIF-1, and Their Inhibitors: An Overview on Pediatric Cancers.

During the past decades, several discoveries have established the role of epigenetic modifications and cellular microenvironment in tumor growth and progression. One of the main representatives concerning epigenetic modification is the polycomb group (PcG). It is composed of different highly conserved epigenetic effector proteins preserving, through several post-translational modifications of histones, the silenced state of the genes implicated in a wide range of central biological events such as development, stem cell formation, and tumor progression. Proteins of the PcG can be divided in polycomb repressive complexes (PRCs): PRC1 and PRC2. In particular, enhancer of zeste homolog 2 (EZH2), the catalytic core subunit of PRC2, acts as an epigenetic silencer of many tumor suppressor genes through the trimethylation of lysine 27 on histone H3, an essential binding site for DNA methyl transferases and histone deacetylases. A growing number of data suggests that overexpression of EZH2 associates with progression and poor outcome in a large number of cancer cases. Hypoxia inducible factor (HIF) is an important transcription factor involved in modulating cellular response to the microenvironment by promoting and regulating tumor development such as angiogenesis, inflammation, metabolic reprogramming, invasion, and metastatic fate. The HIF complex is represented by different subunits (α and ß) acting together and promoting the expression of vascular endothelial growth factor (VEGF), hexokinase II (HKII), receptor for advanced glycation end products (RAGE), carbonic anhydrase (CA), etc., after binding to the hypoxia-response element (HRE) binding site on the DNA. In this review, we will try to connect these two players by detailing the following: (i) the activity and influence of these two important regulators of cancer progression in particular for what concerns pediatric tumors, (ii) the possible correlation between them, and (iii) the feasibility and efficiency to contrast them using several inhibitors.

Notch and NF-κB: Coach and Players of Regulatory T-Cell Response in Cancer.

The Notch signaling pathway plays multiple roles in driving T-cell fate decisions, proliferation, and aberrant growth. NF-κB is a cell-context key player interconnected with Notch signaling either in physiological or in pathological conditions. This review focuses on how the multilayered crosstalk between different Notches and NF-κB subunits may converge on Foxp3 gene regulation and orchestrate CD4+ regulatory T (Treg) cell function, particularly in a tumor microenvironment. Notably, Treg cells may play a pivotal role in the inhibition of antitumor immune responses, possibly promoting tumor growth. A future challenge is represented by further dissection of both Notch and NF-κB pathways and consequences of their intersection in tumor-associated Treg biology. This may shed light on the molecular mechanisms regulating Treg cell expansion and migration to peripheral lymphoid organs thought to facilitate tumor development and still to be explored. In so doing, new opportunities for combined and/or more selective therapeutic approaches to improve anticancer immunity may be found.

Intrathymic Notch3 and CXCR4 combinatorial interplay facilitates T-cell leukemia propagation.

Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying "pre-leukemic" cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4+CD8+ thymocytes, possibly contributing to "pre-leukemic" cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4+CD8+ cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4+CD8+ cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4+CD8+ cells and such an expression is regulated by Notch3 through ß-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4+CD8+ cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4+CD8+ cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of "pre-leukemic" CD4+CD8+ cells, by a "thymus-autonomous" mechanism.

The Notch3 Receptor and Its Intracellular Signaling-Dependent Oncogenic Mechanisms.

During evolution, gene duplication of the Notch receptor suggests a progressive functional diversification. The Notch3 receptor displays a number of structural differences with respect to Notch1 and Notch2, most of which have been reported in the transmembrane and in the intracellular regions, mainly localized in the negative regulatory region (NRR) and trans-activation domain (TAD). Targeted deletion of Notch3 does not result in embryonic lethality, which is in line with its highly restricted tissue expression pattern. Importantly, deregulated Notch3 expression and/or activation, often results in disrupted cell differentiation and/or pathological development, most notably in oncogenesis in different cell contexts. Mechanistically this is due to Notch3-related genetic alterations or epigenetic or posttranslational control mechanisms. In this chapter we discuss the possible relationships between the structural differences and the pathological role of Notch3 in the control of mouse and human cancers. In future, targeting the unique features of Notch3-oncogenic mechanisms could be exploited to develop anticancer therapeutics.

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.