The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Li Cao - One of the best experts on this subject based on the ideXlab platform.
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Wildtype HEK293 Nicastrin amplicon-seq - samtools flagstat
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:These are the samtools flagstat output for an amplicon-seq of HEK293 wildtype cells for a nicatrin amplicon.
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HEK293 Nicastrin heterozygous deletion amplicon-seq - samtools flagstat
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:These data are the samtools flagstat of an amplicon-seq from a HEK293 cell line with heterozygous deletion of Nicastrin.
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HEK293 Nicastrin heterozygous deletion amplicon-seq - target coverage
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:This is the coverage of a Nicastrin amplicon in a HEK293 line with heterozygous deletion of Nicastrin.
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HEK293 Nicastrin knockout RT-qPCR
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:This is an RT-qPCR confirming the knockout (CRISPR-Cas9 mediated) of Nicastrin in HEK293 cells.
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Nicastrin knockdown growth curve data
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:Growth curve data for cell lines knocked down for Nicastrin versus cells knocked down for a control.
Elisha Roberson - One of the best experts on this subject based on the ideXlab platform.
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Wildtype HEK293 Nicastrin amplicon-seq - samtools flagstat
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:These are the samtools flagstat output for an amplicon-seq of HEK293 wildtype cells for a nicatrin amplicon.
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HEK293 Nicastrin heterozygous deletion amplicon-seq - samtools flagstat
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:These data are the samtools flagstat of an amplicon-seq from a HEK293 cell line with heterozygous deletion of Nicastrin.
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HEK293 Nicastrin heterozygous deletion amplicon-seq - target coverage
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:This is the coverage of a Nicastrin amplicon in a HEK293 line with heterozygous deletion of Nicastrin.
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HEK293 Nicastrin knockout RT-qPCR
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:This is an RT-qPCR confirming the knockout (CRISPR-Cas9 mediated) of Nicastrin in HEK293 cells.
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Nicastrin knockdown growth curve data
2019Co-Authors: Elisha Roberson, David Morales-heil, Li CaoAbstract:Growth curve data for cell lines knocked down for Nicastrin versus cells knocked down for a control.
Ylenia Lombardo - One of the best experts on this subject based on the ideXlab platform.
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Endocrine therapy resistance and epithelial to mesenchymal transition are driven by Nicastrin and Notch4 cooperation in MCF7 breast cancer cells.
Cancer Cell & Microenvironment, 2014Co-Authors: Monica Faronato, Ylenia Lombardo, R. Charles CoombesAbstract:Endocrine therapy resistant (ETR) tumors often display mesenchymal features, associated with aggressive and enhanced motility behaviour. Notch signalling is over-activated in ETR cells. By blocking it, it is possible to interfere with the cell growth. Notch is also implicated in regulating epithelial to mesenchymal transition (EMT) affecting both migration and invasion in breast cancer cells. We know that Nicastrin is the major component of the multi-subunit protease complex Gamma Secretase (GS) which executes intramembrane proteolysis of integral proteins such Notch. We used two models of Endocrine Therapy Resistant MCF7 breast cancer cell lines which acquired EMT feature and showed higher levels of Nicastrin and Notch targets. Moreover, they displayed higher Notch4 levels but lower Notch1 and Notch2, indicating a possible switch of the Notch signalling when cells acquire resistance. We demonstrated that Gamma Secretase inhibitor PF03084014 and anti-Nicastrin MAbs were particularly effective in partially inhibiting the EMT process by blocking Nicastrin-Notch4 contribution to resistance. Importantly, we also demonstrated that the stem cells-like population was reduced upon these treatments. Both Nicastrin and Notch4 genetic silencing lead to similar effects. Finally, stably overexpressing Nicastrin, was sufficient to activate Notch4 in MCF7 and render them unresponsive to tamoxifen. Here we highlight our recent study.
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Anti-Nicastrin monoclonal antibodies elicit pleiotropic anti-tumour pharmacological effects in invasive breast cancer cells
Breast cancer research and treatment, 2014Co-Authors: Aleksandra Filipovic, Andrew R. Green, Ylenia Lombardo, Monica Fronato, Joel Abrahams, Eric O. Aboagye, Quang-dé Nguyen, Barbara Borda D’aqua, Anne J. Ridley, Emad RahkaAbstract:The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, Nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-Nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed Nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of Nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic.
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Nicastrin and notch4 drive endocrine therapy resistance and epithelial to mesenchymal transition in mcf7 breast cancer cells
Breast Cancer Research, 2014Co-Authors: Ylenia Lombardo, Monica Faronato, Aleksandra Filipovic, Valentina Vircillo, Luca Magnani, Charles R CoombesAbstract:Resistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ERα)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells. We used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen. We found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation. ETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.
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Abstract 4762: Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer .
Endocrinology, 2013Co-Authors: Monica Faronato, Ylenia Lombardo, Aleksandra Filipovic, Simak Ali, R. Charles CoombesAbstract:Background: Resistance to endocrine therapy occurs in at least 30% of patients with breast cancer (BC) and remains a major clinical problem. Among the mechanisms underpinning endocrine resistance is the enrichment of epithelial-to-mesenchymal transition (EMT) features and signalling pathways such as EGFR and Notch. Notch receptors are activated by the gamma-secretase (GS) enzyme composed of Nicastrin, presenilin, PEN2 and Aph-1. Gamma secretase inhibitors are being tested as novel therapeutics for cancer treatment, predominantly in combination with anti-hormonal agents as well as chemotherapy, however treatment related toxicities are apparent. Nicastrin is upregulated in BC and we have developed anti-Nicastrin monoclonal antibodies (MAbs) to target its functions and block GS. Here, we show that Nicastrin may be a relevant therapeutic target in endocrine resistance and demonstrate the effects of anti-Nicastrin MAbs. Materials and Methods: We used two cell line models of MCF7 cells which developed endocrine resistance: TamR cells (Hiscox et al) and MLET2/MLET5 developed in our laboratory. We raised anti-Nicastrin MAbs (MAb1 and MAb2) against Nicastrin extracellular domain. Western blotting, RT-qPCR, transwell invasion and 3D matrigel growth assays were used to demonstrate their molecular and functional effects. Gamma secreatse inhibitor RO4929097 was used for comparison. Results: We demonstrate that Nicastrin is overexpressed at both mRNA and protein level in endocrine resistant cell lines (> 4-fold). These cells concomitantly exhibit elevated molecular markers of EMT: Vimentin (3-fold), CD44 (> 2-fold), EGFR (> 4-fold), RhoGTPase Cdc42 target genes IQGAP1 (2-fold) and IQGAP2 (>100-fold); as well as Notch4 (>2-fold), Notch-targets Hey1 and Hes1 (4-fold), while Notch1 is suppressed (> 2-fold). Anti-Nicastrin MAb1, MAb2 and RO4929097 were equally potent in inhibiting Notch target genes Hey1 and Hes1, as well as growth of TAMR and MLET acini in 3D Matrigel. Interestingly, effective inhibition of vimentin, CD44 and IQGAPs was observed only upon treatment with anti-Nicastrin MAbs, while RO4929097 had no effect. Interestinly, a clear distinction was noted between anti-Nicastrin MAbs and RO4929097 in terms of their effect on Notch4. MAb1 and MAb2 attenuated Notch4 transcription and reduced activated Notch4 and Nicastrin levels, while RO4929097 achieved an opposite effect. Furthermore, only anti-Nicastrin MAbs were also potent inhibitors of TAMR cell invasion (by >50%, p Conclusion: Nicastrin is overexpressed in endocrine resistant breast cancer cells. Anti-Nicastrin monoclonal antibodies inhibit Notch4 signalling, attenuate expression of pro-invasive molecules such as vimentin, IQGAPs and CD44 and reduce cellular invasiveness and growth in 3D Matrigel. Therefore, targeting Nicastrin may be a valid therapeutic strategy in breast cancer patients that relapse of endocrine therapy. Citation Format: Monica Faronato, Ylenia Lombardo, Aleksandra Filipovic, Simak Ali, R.Charles Coombes. Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4762. doi:10.1158/1538-7445.AM2013-4762
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Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo.
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Ylenia Lombardo, Jayson Wang, Ernesto Yagüe, Aleksandra Filipovic, Gemma Molyneux, Manikandan Periyasamy, Georgios Giamas, Pritesh Trivedi, Loren S. MichelAbstract:Nicastrin (NCT) is a crucial component of the γ-secretase (GS) enzyme, which prompted investigations into its biological role in cancer. We have previously shown that Nicastrin is overexpressed in breast cancer (BC), conferring worse overall survival in invasive, ERα negative patients. Here, we used 2D and 3D Matrigel, anchorage-independent growth conditions and a breast cancer xenograft mouse model to assess the impact of Nicastrin on breast cancer stem cell (BCSC) propagation and invasion in vitro and tumor growth in vivo. Stable knockdown of Nicastrin in HCC1806 breast cancer cells reduced cell invasion by 51.4 ± 1.7%, accompanied by a morphological change to a rounded cell phenotype and down-regulation of vimentin, Snail, Twist, MMP2, and MMP9. We observed a reduction of the pool of CD44(+)/CD24(-) and ALDH1 high breast cancer stem cells by threefold and twofold, respectively, and a reduction by 2.6-fold of the mammospheres formation. Nicastrin overexpression in nontransformed MCF10A cells caused an induction of epithelial to mesenchymal regulators, as well as a fivefold increased ALDH1 activity, a threefold enrichment for CD44(+)/CD24(-) stem cells, and a 3.2-fold enhanced mammosphere-forming capacity. Using the γ-sescretase inhibiton, Notch1/4 siRNA, and Akt inhibition, we show that Nicastrin regulates breast cancer stem cells partly through Notch1 and the Akt pathway. Exploiting serial dilution transplantation of the HCC1806 cells expressing Nicastrin and HCC1806 stably depleted of Nicastrin, in vivo, we demonstrate that Nicastrin inhibition may be relevant for the reduced tumorigenicity of breast cancer cells. These data could serve as a benchmark for development of Nicastrin-targeted therapies in breast cancer.
Bart De Strooper - One of the best experts on this subject based on the ideXlab platform.
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Glu332 in the Nicastrin Ectodomain Is Essential for γ-Secretase Complex Maturation but Not for Its Activity
The Journal of biological chemistry, 2008Co-Authors: Lucía Chávez-gutiérrez, Philip C. Wong, Alexandra Tolia, Elke Maes, Bart De StrooperAbstract:Abstract The γ-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu333 in human) serving as the anionic binding site for the α-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu332 is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu332 could bind the α-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic α-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased γ-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu332 crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally γ-secretase activity. Consequently, these studies indicate that the main role of the Glu332 is in the maturation and assembly of γ-secretase rather than in the recognition of the substrates.
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Nicastrin: Gatekeeper of the γ-Secretase Complex
Cell, 2005Co-Authors: Bart De StrooperAbstract:The gamma-secretase intramembrane protease cleaves many type I membrane proteins including amyloid precursor protein and Notch, generating peptide fragments that are important signaling components. In this issue of Cell, Shah et al. (2005) reveal the function of Nicastrin, the largest member of the gamma-secretase complex. They show that the Nicastrin extracellular domain is essential for recognition of substrate by the gamma-secretase.
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presenilins mutated at asp 257 or asp 385 restore pen 2 expression and Nicastrin glycosylation but remain catalytically inactive in the absence of wild type presenilin
Journal of Biological Chemistry, 2003Co-Authors: Omar Nyabi, A. Herreman, Mostafa Bentahir, Katrien Horré, Christine Van Broeckhoven, Pascal Merchiers, Kurt Spittaels, Wim Annaert, Numa R Gottardilittell, Bart De StrooperAbstract:The Presenilins are part of the γ-secretase complex that is involved in the regulated intramembrane proteolysis of amyloid precursor protein and other type I integral membrane proteins. Nicastrin, Pen-2, and Aph1 are the other proteins of this complex. The Presenilins probably contribute the catalytic activity to the protease complex. However, several investigators reported normal Aβ-peptide generation in cells expressing Presenilins mutated at the putative catalytic site residue Asp-257, contradicting this hypothesis. Because endogenously expressed wild type Presenilin could contribute to residual γ-secretase activity in these experiments, we have reinvestigated the problem by expressing mutated Presenilins in a Presenilin-negative cell line. We confirm that Presenilins with mutated Asp residues are catalytically inactive. Unexpectedly, these mutated Presenilins are still partially processed into amino- and carboxyl-terminal fragments by a "Presenilinase"-like activity. They are also able to rescue Pen-2 expression and Nicastrin glycosylation in Presenilin-negative cells and become incorporated into large ∼440-kDa complexes as assessed by blue native gel electrophoresis. Our study demonstrates that the catalytic activity of Presenilin and its other functions in the generation, stabilization, and transport of the γ-secretase complex can be separated and extends the concept that Presenilins are multifunctional proteins.
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Presenilins mutated at Asp-257 or Asp-385 restore Pen-2 expression and Nicastrin glycosylation but remain catalytically inactive in the absence of wild type Presenilin.
The Journal of biological chemistry, 2003Co-Authors: Omar Nyabi, A. Herreman, Mostafa Bentahir, Katrien Horré, Numa R. Gottardi-littell, Christine Van Broeckhoven, Pascal Merchiers, Kurt Spittaels, Wim Annaert, Bart De StrooperAbstract:The Presenilins are part of the gamma-secretase complex that is involved in the regulated intramembrane proteolysis of amyloid precursor protein and other type I integral membrane proteins. Nicastrin, Pen-2, and Aph1 are the other proteins of this complex. The Presenilins probably contribute the catalytic activity to the protease complex. However, several investigators reported normal Abeta-peptide generation in cells expressing Presenilins mutated at the putative catalytic site residue Asp-257, contradicting this hypothesis. Because endogenously expressed wild type Presenilin could contribute to residual gamma-secretase activity in these experiments, we have reinvestigated the problem by expressing mutated Presenilins in a Presenilin-negative cell line. We confirm that Presenilins with mutated Asp residues are catalytically inactive. Unexpectedly, these mutated Presenilins are still partially processed into amino- and carboxyl-terminal fragments by a "Presenilinase"-like activity. They are also able to rescue Pen-2 expression and Nicastrin glycosylation in Presenilin-negative cells and become incorporated into large approximately 440-kDa complexes as assessed by blue native gel electrophoresis. Our study demonstrates that the catalytic activity of Presenilin and its other functions in the generation, stabilization, and transport of the gamma-secretase complex can be separated and extends the concept that Presenilins are multifunctional proteins.
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gamma secretase activity requires the presenilin dependent trafficking of Nicastrin through the golgi apparatus but not its complex glycosylation
Journal of Cell Science, 2003Co-Authors: A. Herreman, Omar Nyabi, Mostafa Bentahir, Wim Annaert, Geert Van Gassen, Katleen Craessaerts, Ulrike Mueller, Bart De StrooperAbstract:Nicastrin and presenilin are two major components of the γ-secretase complex, which executes the intramembrane proteolysis of type I integral membrane proteins such as the amyloid precursor protein (APP) and Notch. Nicastrin is synthesized in fibroblasts and neurons as an endoglycosidase-H-sensitive glycosylated precursor protein (immature Nicastrin) and is then modified by complex glycosylation in the Golgi apparatus and by sialylation in the trans-Golgi network (mature Nicastrin). These modifications are not observed with exogenously overexpressed Nicastrin. Under normal cell culture conditions, only mature Nicastrin is expressed at the cell surface and binds to the presenilin heterodimers. Mature Nicastrin has a half-life of more than 24 hours. In the absence of presenilin 1 and 2, Nicastrin remains entirely endoglycosidase H sensitive, is retained in the endoplasmic reticulum and is slowly degraded. Single presenilin 1 or presenilin 2 deficiency affects glycosylation of Nicastrin to a lesser extent than the combined presenilin deficiencies, suggesting a correlation between either the transport of Nicastrin out of the endoplasmic reticulum or the concomitant complex glycosylation of Nicastrin, and γ-secretase activity. However, when complex glycosylation of Nicastrin was inhibited using mannosidase I inhibitors, γ-secretase cleavage of APP or Notch was not inhibited and the immature Nicastrin still associates with presenilin and appears at the cell surface. Complex glycosylation of Nicastrin is therefore not needed for γ-secretase activity. Because the trafficking of Nicastrin to the Golgi apparatus is dependent on presenilins, our data point to a central role of presenilin in Nicastrin maturation/localization, which could help to partially resolve the `spatial paradox9.
Aleksandra Filipovic - One of the best experts on this subject based on the ideXlab platform.
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Anti-Nicastrin monoclonal antibodies elicit pleiotropic anti-tumour pharmacological effects in invasive breast cancer cells
Breast cancer research and treatment, 2014Co-Authors: Aleksandra Filipovic, Andrew R. Green, Ylenia Lombardo, Monica Fronato, Joel Abrahams, Eric O. Aboagye, Quang-dé Nguyen, Barbara Borda D’aqua, Anne J. Ridley, Emad RahkaAbstract:The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, Nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-Nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed Nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of Nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic.
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Nicastrin and notch4 drive endocrine therapy resistance and epithelial to mesenchymal transition in mcf7 breast cancer cells
Breast Cancer Research, 2014Co-Authors: Ylenia Lombardo, Monica Faronato, Aleksandra Filipovic, Valentina Vircillo, Luca Magnani, Charles R CoombesAbstract:Resistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ERα)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells. We used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen. We found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation. ETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.
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The role of genes co-amplified with Nicastrin in breast invasive carcinoma
Breast cancer research and treatment, 2013Co-Authors: Anida Sarajlić, Rc Coombes, Aleksandra Filipovic, Vuk Janjić, Nataša PržuljAbstract:Breast cancer accounts for more than 450,000 deaths per year worldwide. Discovery of novel therapeutic targets that will allow patient-tailored treatment of this disease is an emerging area of scientific interest. Recently, Nicastrin has been identified as one such therapeutic target. Its overexpression is indicative of worse overall survival in the estrogen-receptor-negative patient population. In this paper, we analyze data from a large invasive breast carcinoma study and confirm Nicastrin amplification. In search for genes that are co-amplified with Nicastrin, we identify a potential novel breast cancer-related amplicon located on chromosome 1. Furthermore, we search for “influential interactors,” i.e., genes that interact with a statistically significantly high number of genes which are co-amplified with Nicastrin, and confirm their involvement in this female neoplasm. Among the influential interactors, we find genes which belong to the core diseasome (a recently identified therapeutically relevant set of genes which is known to drive disease formation) and propose that they might be important for breast cancer onset, and serve as its novel therapeutic targets. Finally, we identify a pathway that may play a role in Nicastrin’s amplification process and we experimentally confirm downstream signaling mechanism of Nicastrin in breast cancer cells.
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Abstract 4762: Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer .
Endocrinology, 2013Co-Authors: Monica Faronato, Ylenia Lombardo, Aleksandra Filipovic, Simak Ali, R. Charles CoombesAbstract:Background: Resistance to endocrine therapy occurs in at least 30% of patients with breast cancer (BC) and remains a major clinical problem. Among the mechanisms underpinning endocrine resistance is the enrichment of epithelial-to-mesenchymal transition (EMT) features and signalling pathways such as EGFR and Notch. Notch receptors are activated by the gamma-secretase (GS) enzyme composed of Nicastrin, presenilin, PEN2 and Aph-1. Gamma secretase inhibitors are being tested as novel therapeutics for cancer treatment, predominantly in combination with anti-hormonal agents as well as chemotherapy, however treatment related toxicities are apparent. Nicastrin is upregulated in BC and we have developed anti-Nicastrin monoclonal antibodies (MAbs) to target its functions and block GS. Here, we show that Nicastrin may be a relevant therapeutic target in endocrine resistance and demonstrate the effects of anti-Nicastrin MAbs. Materials and Methods: We used two cell line models of MCF7 cells which developed endocrine resistance: TamR cells (Hiscox et al) and MLET2/MLET5 developed in our laboratory. We raised anti-Nicastrin MAbs (MAb1 and MAb2) against Nicastrin extracellular domain. Western blotting, RT-qPCR, transwell invasion and 3D matrigel growth assays were used to demonstrate their molecular and functional effects. Gamma secreatse inhibitor RO4929097 was used for comparison. Results: We demonstrate that Nicastrin is overexpressed at both mRNA and protein level in endocrine resistant cell lines (> 4-fold). These cells concomitantly exhibit elevated molecular markers of EMT: Vimentin (3-fold), CD44 (> 2-fold), EGFR (> 4-fold), RhoGTPase Cdc42 target genes IQGAP1 (2-fold) and IQGAP2 (>100-fold); as well as Notch4 (>2-fold), Notch-targets Hey1 and Hes1 (4-fold), while Notch1 is suppressed (> 2-fold). Anti-Nicastrin MAb1, MAb2 and RO4929097 were equally potent in inhibiting Notch target genes Hey1 and Hes1, as well as growth of TAMR and MLET acini in 3D Matrigel. Interestingly, effective inhibition of vimentin, CD44 and IQGAPs was observed only upon treatment with anti-Nicastrin MAbs, while RO4929097 had no effect. Interestinly, a clear distinction was noted between anti-Nicastrin MAbs and RO4929097 in terms of their effect on Notch4. MAb1 and MAb2 attenuated Notch4 transcription and reduced activated Notch4 and Nicastrin levels, while RO4929097 achieved an opposite effect. Furthermore, only anti-Nicastrin MAbs were also potent inhibitors of TAMR cell invasion (by >50%, p Conclusion: Nicastrin is overexpressed in endocrine resistant breast cancer cells. Anti-Nicastrin monoclonal antibodies inhibit Notch4 signalling, attenuate expression of pro-invasive molecules such as vimentin, IQGAPs and CD44 and reduce cellular invasiveness and growth in 3D Matrigel. Therefore, targeting Nicastrin may be a valid therapeutic strategy in breast cancer patients that relapse of endocrine therapy. Citation Format: Monica Faronato, Ylenia Lombardo, Aleksandra Filipovic, Simak Ali, R.Charles Coombes. Anti-Nicastrin antibodies for the treatment of endocrine resistant breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4762. doi:10.1158/1538-7445.AM2013-4762
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Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo.
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Ylenia Lombardo, Jayson Wang, Ernesto Yagüe, Aleksandra Filipovic, Gemma Molyneux, Manikandan Periyasamy, Georgios Giamas, Pritesh Trivedi, Loren S. MichelAbstract:Nicastrin (NCT) is a crucial component of the γ-secretase (GS) enzyme, which prompted investigations into its biological role in cancer. We have previously shown that Nicastrin is overexpressed in breast cancer (BC), conferring worse overall survival in invasive, ERα negative patients. Here, we used 2D and 3D Matrigel, anchorage-independent growth conditions and a breast cancer xenograft mouse model to assess the impact of Nicastrin on breast cancer stem cell (BCSC) propagation and invasion in vitro and tumor growth in vivo. Stable knockdown of Nicastrin in HCC1806 breast cancer cells reduced cell invasion by 51.4 ± 1.7%, accompanied by a morphological change to a rounded cell phenotype and down-regulation of vimentin, Snail, Twist, MMP2, and MMP9. We observed a reduction of the pool of CD44(+)/CD24(-) and ALDH1 high breast cancer stem cells by threefold and twofold, respectively, and a reduction by 2.6-fold of the mammospheres formation. Nicastrin overexpression in nontransformed MCF10A cells caused an induction of epithelial to mesenchymal regulators, as well as a fivefold increased ALDH1 activity, a threefold enrichment for CD44(+)/CD24(-) stem cells, and a 3.2-fold enhanced mammosphere-forming capacity. Using the γ-sescretase inhibiton, Notch1/4 siRNA, and Akt inhibition, we show that Nicastrin regulates breast cancer stem cells partly through Notch1 and the Akt pathway. Exploiting serial dilution transplantation of the HCC1806 cells expressing Nicastrin and HCC1806 stably depleted of Nicastrin, in vivo, we demonstrate that Nicastrin inhibition may be relevant for the reduced tumorigenicity of breast cancer cells. These data could serve as a benchmark for development of Nicastrin-targeted therapies in breast cancer.