Search

N-myristoyltransferases (NMTs) catalyze the protein N-terminal modification N-myristoylation, a lipidation event which affects >150 proteins and is involved in protein localization, stability, and function. NMT has been suggested as a target in cancers, but there has been a lack of rationale to identify patients who may respond to NMT inhibition. Additionally, the mechanism of action of NMT inhibitors (NMTi) is difficult to dissect, as NMT substrates feature in multiple biological pathways, and many studies have focused only on a single substrate. Here, a combination of bioinformatic, biochemical, proteomic, and cellular biology techniques have been combined to show that deregulation of MYC-family proteins sensitizes cells to NMT inhibition. Cell lines sensitive to NMTi were identified through screening of hundreds of cancer cell lines, and the transcriptome of sensitive and insensitive lines compared to obtain a “Sensitive to NMTi” gene set. This gene set correlated well with MYC-related gene sets and mutation, amplification, or chromosomal rearrangement in MYC(N) were predictive for NMTi sensitivity, suggesting that MYC deregulated cancer cells are sensitive to NMTi. This was verified by cytotoxicity assays in B-cell lymphoma and neuroblastoma lines, where highly MYC(N) expressing lines exhibited increased NMTi sensitivity, including in the SHEP21N and P493-6 lines, in which MYC(N) levels can be regulated. Furthermore, proteomic profiling identified multiple pathways which are affected by NMTi, and these were further validated. In particular, an impact on Complex I formation was seen, and mitochondrial dysfunction in high MYC(N) cells upon NMTi was seen through the loss of basal and maximal respiration, ATP production and spare respiratory capacity. Loss of Complex I formation was shown to be caused by the loss of the NMT substrate NDUFAF4 upon NMTi treatment. Furthermore, application of an orally bioavailable NMTi eliminated tumors in mouse models of both diffuse large B-cell lymphoma and neuroblastoma and were well tolerated with no changed in body weight, suggesting that NMT inhibitors are well tolerated and efficacious in vivo. Citation Format: James Zhang, Gregor A. Lueg, Monica Faronato, Evon Poon, Andrii Gorelik, Andrea G. Grocin, Eva Caamano-Gutierrez, Francesco Falciani, Roberto Solari, Robin Carr, Andrew S. Bell, Edward Bartlett, Jennie Hutton, Miriam Llorian-Sopena, Probir Chakravarty, Bernadette Brzezicha, Martin Janz, Matther J. Garnett, Louis Chesler, Dinis P. Calado, Edward W. Tate. Dysregulation of MYC-family proteins sensitizes cancers to NMT inhibition: identification of NMTi sensitivity and mechanism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4871.

Human N-myristoyltransferases (NMTs) catalyze N-terminal protein myristoylation, a modification regulating membrane trafficking and interactions of >100 proteins. NMT is a promising target in cancer, but a mechanistic rationale for targeted therapy remains poorly defined. Here, large-scale cancer cell line screens against a panel of NMT inhibitors (NMTi) were combined with systems-level analyses to reveal that NMTi is synthetic lethal with deregulated MYC. Synthetic lethality is mediated by post-transcriptional failure in mitochondrial respiratory complex I protein synthesis concurrent with loss of myristoylation and degradation of complex I assembly factor NDUFAF4, followed by mitochondrial dysfunction specifically in MYC-deregulated cancer cells. NMTi eliminated MYC-deregulated tumors in vivo without overt toxicity, providing a new paradigm in which targeting a constitutive co-translational protein modification is synthetically lethal in MYC-deregulated cancers.One-sentence summaryN-myristoyltransferase inhibition leads to post-transcriptional complex I failure and cell death in MYC-deregulated cancers

Approximately 30% of ERα breast cancer patients relapse with metastatic disease following adjuvant endocrine therapies. The connection between acquisition of drug resistance and invasive potential is poorly understood. In this study, we demonstrate that the type II keratin topological associating domain undergoes epigenetic reprogramming in aromatase inhibitors (AI)-resistant cells, leading to Keratin-80 (KRT80) upregulation. KRT80 expression is driven by de novo enhancer activation by sterol regulatory element-binding protein 1 (SREBP1). KRT80 upregulation directly promotes cytoskeletal rearrangements at the leading edge, increased focal adhesion and cellular stiffening, collectively promoting cancer cell invasion. Shearwave elasticity imaging performed on prospectively recruited patients confirms KRT80 levels correlate with stiffer tumors. Immunohistochemistry showed increased KRT80-positive cells at relapse and, using several clinical endpoints, KRT80 expression associates with poor survival. Collectively, our data uncover an unpredicted and potentially targetable direct link between epigenetic and cytoskeletal reprogramming promoting cell invasion in response to chronic AI treatment.

Human N-myristoyltransferase (NMT) 1 and 2 catalyze N-terminal protein myristoylation, a modification that regulates membrane trafficking and interactions of >100 proteins. NMT has been proposed as a target in cancer, but a rationale for selectivity is lacking due to the complex impact of NMT inhibition (NMTi) on multiple cellular pathways. Here, large-scale screens of hundreds of cancer cell lines against a panel of potent and selective NMTi were combined with systems-level analyses to reveal that NMTi is synthetically lethal with deregulated MYC or MYCN. Synthetic lethality is mediated by post-transcriptional failure in (NADH) Complex I protein synthesis followed by mitochondrial dysfunction in MYC-deregulated cancer cells. We observed that NMT inhibitors blocked the growth of patient-derived tumors with deregulated MYC, without overt toxicity. This mechanistic framework supports NMTi as a novel targeted therapeutic approach and provides a new paradigm in which targeting of a constitutive protein modification is synthetically lethal in MYC-deregulated cancers. Citation Format: Gregor A. Lueg, Monica Faronato, Andrii Gorelik, Andrea G. Grocin, Miriam Llorian-Sopena, Probir Chakravarty, Bernadette Brzezicha, Martin Janz, Mathew J. Garnett, Dinis P. Calado, Edward W. Tate. Targeting MYC deregulation in cancer [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA30.

Summary On-target, cell-active chemical probes are of fundamental importance in chemical and cell biology, whereas poorly characterized probes often lead to invalid conclusions. Human N-myristoyltransferase (NMT) has attracted increasing interest as target in cancer and infectious diseases. Here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis, and cell-cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM), or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies that used off-target compounds., Graphical Abstract, Highlights • IMP-1088 selectively blocks N-myristoylation in cells at low nanomolar concentration • Three widely used human NMT probes are invalidated by 6 orthogonal assay modalities • 2-Hydroxymyristate is inactive up to 100 μM; D-NMAPPD is non-specifically cytotoxic • Tris-DBA palladium does not inhibit NMT in cells, and is cytotoxic by precipitation, Specific inhibitors of protein N-myristoylation are powerful probes for exploring the role of this post-translational modification in cells and have therapeutic potential. Kallemeijn et al. identify three flawed but commonly applied human N-myristoyltransferase inhibitors, and advocate two classes of high-affinity inhibitors as probes with a well-validated on-target cellular mode of action.

Approximately 30% of women diagnosed with ERα breast cancer relapse with metastatic disease following adjuvant treatment with endocrine therapies1,2. The connection between acquisition of drug resistance and invasive potential is poorly understood. In this study, we demonstrate that the type II keratin topological associating domain (TAD)3 undergoes epigenetic reprogramming in cells that develop resistance to aromatase inhibitors (AI), leading to keratin 80 (KRT80) upregulation. In agreement, an increased number of KRT80-positive cells are observed at relapse in vivo while KRT80 expression associates with poor outcome using several clinical endpoints. KRT80 expression is driven by de novo enhancer activation by sterol regulatory element-binding protein 14 (SREBP1). KRT80 upregulation directly promotes cytoskeletal rearrangements at the leading edge, increased focal adhesion maturation and cellular stiffening, which collectively promote cancer cell invasion. Shear-wave elasticity imaging of prospective patients shows that KRT80 levels correlate with stiffer tumors in vivo. Collectively, our data uncover an unpredicted and potentially targetable direct link between epigenetic and cytoskeletal reprogramming promoting cell invasion in response to chronic AI treatment.

Post-translational attachment of lipids to proteins is found in all organisms, and is important for many biological processes. Acylation with myristic and palmitic acids are among the most common lipid modifications, and understanding reversible protein palmitoylation dynamics has become a particularly important goal. Linking acyltransferase enzymes to disease states can be challenging due to a paucity of robust models, compounded by functional redundancy between many palmitoyl transferases; however, in cases such as Wnt or Hedgehog signalling, small molecule inhibitors have been identified, with some progressing to clinical trials. In this review, we present recent developments in our understanding of protein acylation in human health and disease through use of chemical tools, global profiling of acylated proteomes, and functional studies of specific protein targets.

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients.

Reversible ubiquitylation of proteins contributes to their integrity, abundance and activity. The RE1-silencing transcription factor (REST) plays key physiological roles and is dysregulated in a spectrum of disease. It is rapidly turned over and is phosphorylated, polyubiquitylated and degraded en masse during neuronal differentiation and cell cycle progression. Through siRNA screening we identified the deubiquitylase USP15 as a key regulator of cellular REST. Both antagonism of REST polyubiquitylation and rescue of endogenous REST levels are dependent on the deubiquitylase activity of USP15. However, USP15 depletion does not destabilize pre-existing REST, but rather specifically impairs de novo REST synthesis. Indeed, we find that a small fraction of endogenous USP15 is associated with polysomes. In accordance with these findings, USP15 does not antagonize the degradation of phosphorylated REST at mitosis. Instead it is required for the rapid accumulation of newly synthesized REST on mitotic exit, thus playing a key role in its cell cycle oscillations. Importantly, this study reveals a novel role for a DUB in specifically promoting new protein synthesis.

Summary The scattering response of epithelial cells to activation of the Met receptor tyrosine kinase represents one facet of an “invasive growth” program [1, 2]. It is a complex event that incorporates loss of cell-cell adhesion, morphological changes, and cell motility. Ubiquitination is a reversible posttranslational modification that may target proteins for degradation or coordinate signal transduction pathways [3, 4]. There are ∼79 active deubiquitinating enzymes (DUBs) predicted in the human genome [5, 6]. Here, via a small interfering RNA (siRNA) library approach, we have identified 12 DUBs that are necessary for aspects of the hepatocyte growth factor (HGF)-dependent scattering response of A549 cells. Different phenotypes are evident that range from full loss of scattering, similar to receptor knockdown (e.g., USP30, USP33, USP47), to loss of cell-cell contacts even in the absence of HGF but defective motility (e.g., USP3, ATXN3L). The knockdowns do not incur defective receptor, phosphatidylinositol 3-kinase, or MAP kinase activation. Our data suggest widespread involvement of the ubiquitin system at multiple stages of the Met activation response, implying significant crosstalk with phosphorylation-based transduction pathways. Development of small-molecule inhibitors of particular DUBs may offer a therapeutic approach to contain metastasis.

Endocrine therapies target the activation of the oestrogen receptor alpha (ERα) via distinct mechanisms, but it is not clear whether breast cancer cells can adapt to treatment using drug-specific mechanisms. Here we demonstrate that resistance emerges via drug-specific epigenetic reprogramming. Resistant cells display a spectrum of phenotypical changes with invasive phenotypes evolving in lines resistant to the aromatase inhibitor (AI). Orthogonal genomics analysis of reprogrammed regulatory regions identifies individual drug-induced epigenetic states involving large topologically associating domains (TADs) and the activation of super-enhancers. AI-resistant cells activate endogenous cholesterol biosynthesis (CB) through stable epigenetic activation in vitro and in vivo. Mechanistically, CB sparks the constitutive activation of oestrogen receptors alpha (ERα) in AI-resistant cells, partly via the biosynthesis of 27-hydroxycholesterol. By targeting CB using statins, ERα binding is reduced and cell invasion is prevented. Epigenomic-led stratification can predict resistance to AI in a subset of ERα-positive patients., How breast cancer cells adapt to individual therapies targeting the oestrogen receptor alpha is poorly understood. Here the authors show resistance emerging through differential epigenetic reprogramming that activates the cholesterol biosynthesis pathway.

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.

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. Electronic supplementary material The online version of this article (doi:10.1007/s10549-014-3119-z) contains supplementary material, which is available to authorized users.

Review on USP15 (ubiquitin specific peptidase 15), with data on DNA, on the protein encoded, and where the gene is implicated.

Review on REST (RE1-silencing transcription factor), with data on DNA, on the protein encoded, and where the gene is implicated.

Given the pivotal role of ion channels in neoplastic transformation, the aim of the present study has been to assess possible differences in the expression patterns of voltage-gated monovalent cationic (Na(+) and K(+)) currents between normal and neoplastic mesothelial cells (NM, MPM, respectively), and to evaluate the role of specific ion channels in mesothelioma cells proliferation, apoptosis, and motility. To achieve this aim, membrane currents expressed in NM and MPM cells derived from surgically-removed human specimens were investigated by means of patch-clamp electrophysiology. NM cells were found to express three main classes of K(+) currents, which were defined as K(IR), maxiK(Ca), and K(V) currents on the basis of their biophysical and pharmacological properties. Each of these K(+) currents was absent in MPM cells; by contrast, MPM cells revealed the novel appearance of tetrodotoxin (TTX)-sensitive voltage-gated Na(+) currents undetected in normal mesothelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR analysis of MPM cells transcripts showed significant expression of the mRNAs encoding for Na(V)1.2, and Na(V)1.6, and Na(V)1.7 (and less so for Na(V)1.3, Na(V)1.4, and Na(V)1.5) main voltage-gated sodium channel (VGSC) alpha-subunit(s). Interestingly, blockade of VGSCs with TTX decreased mesothelioma cell migration in in vitro motility assays; on the other hand, TTX failed to interfere with cell viability, proliferation, and apoptosis progression triggered by UV exposure. In summary, the results of the present study suggest that VGSCs expression in MPM cells may favor the increased motility of the neoplastic cells, a phenotypic feature often associated with the malignant phenotype.

An important aspect of tumor progression is the ability of cancer cells to escape detection and clearance by the immune system. Recent studies suggest that several tumors express soluble factors interfering with the immune response. Here, we show that semaphorin-3A (Sema-3A), a secreted member of the semaphorin family involved in axonal guidance, organogenesis, and angiogenesis, is highly expressed in several tumor cells. Conditioned media of Sema-3A-transfected COS-7 cells or human recombinant Sema-3A inhibited primary human T-cell proliferation and cytokines production under anti-CD3 plus anti-CD28 stimulating conditions. Sema-3A also inhibited the activation of nonspecific cytotoxic activity in mixed lymphocyte culture (MLC), as measured against K-562 cells. In contrast, suppression of Sema-3A in tumor cells with a small interfering RNA (siRNA) augmented T-cell activation. The inhibitory effect of Sema-3A in T cells is mediated by blockade of Ras/mitogen-activated protein kinase (MAPK) signaling pathway. The presence of Sema-3A increased the activation of the Ras family small GTPase Rap1 and introduction of the dominant-negative mutant of Rap1 (Rap1N17) blunted the immunoinhibitory effects of Sema-3A. These results suggest that Sema-3A inhibits primary T-cell activation and imply that it can contribute to the T-cell dysfunction in the tumor microenvironment.

Reference EPFL-ARTICLE-204084doi:10.1007/s10549-014-3174-5View record in Web of Science Record created on 2014-12-30, modified on 2017-12-10

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

Background: Nicastrin (NCT) is the gate keeper of the gamma secretase (GS) enzyme complex. We have previously shown that high levels of NCT are observed in breast cancer patients. Moreover, we recently highlighted the importance of NCT expression for the expansion of breast cancer stem cells and their invasive features. The involvement of NCT in development of chemoresistance has been suggested by the evidence that 1) NCT is increased in response to oxaliplatin, irinotecan and 5-FU in colon cancer and 2) NCT regulates cell death within the p53 and AKT signalling pathway in human embryonic kidney (HEK293) cells. Here, we have explored the potential role of NCT in the chemoresistance of breast cancer cells. Methods: Stable NCT overexpressing MCF10A cells and control cells were produced by retroviral infections. siRNA strategy was used to silence NCT expression in a panel of p53wt and p53mut breast cancer cell lines. The sulphorhodamine B and Annexin V/PI staining were used to measure proliferation and apoptosis rate in breast cancer cells unstressed or treated with chemotherapeutic agents alone or in combination with DAPT, Wortmannin or Nutlin. p53 levels and activity were assessed by Western blotting, RT-qPCR and gene reporter assays. Results: Stable overexpression of NCT in MCF10A cells induced resistance to doxorubicin and cisplatin treatments as demonstrated by a decreased early and late apoptotis. Moreover, NCT lowers p53 expression, its phosphorylation at the phosphoSer15 site and its transcriptional activity in both unstressed cells and doxorubicin-treated cells. Using DAPT and Wortmannin as pharmacological inhibitors of Notch and AKT signalling pathways and Nutlin to inhibit Mdm2-mediated p53 degradation, we confirmed that NCT mediated chemoresistance in a p53 dependent manner partially through the PI3K/AKT pathways, while it is role is completely independent from GS activity. NCT knockdown was able to sensitize several breast cancer cell lines (MCF7, T47D, ZR75) with a wild type p53 status to doxorubicin. Moreover, NCT overexpression reduced the sensitivity to cisplatin in p53wt HTC116 colon cancer cells while it had no effect on p53mut cells. In order to translate these results to clinical settings, we have developed anti-NCT monoclonal antibodies which have been showed to inhibit proliferation and invasion of breast cancer cells. These antibodies used in combination with chemotherapy mirrored the positive effect of NCT siRNA in terms of response to doxorubicin in p53wt breast cancer cell lines. Conclusions: Our study indicates that in breast cancer cells NCT controls chemotherapy-induced cell death in a p53-dependent manner through the regulation of AKT pathway. This function is independent from the GS. We aim to investigate the association of NCT expression and p53 mutation status with clinical outcome in breast cancers in order to support potential combination of anti-NCT monoclonal antibodies with chemotherapy. Citation Format: Ylenia Lombardo, Aleksandra Filipovic, Monica Faronato, Raoul Charles Coombes. Role of nicastrin in the mediating chemoresistance of breast cancer cells. [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 829. doi:10.1158/1538-7445.AM2013-829

Introduction 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. Methods 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. Results 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. Conclusions 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.

// Monica Faronato 1 , Van T.M. Nguyen 1 , Darren K. Patten 1 , Ylenia Lombardo 1 , Jennifer H. Steel 1 , Naina Patel 1 , Laura Woodley 1 , Sami Shousha 1 , Giancarlo Pruneri 2 , R. Charles Coombes 1 , Luca Magnani 1 1 Department of Surgery and Cancer, Imperial College London, London, UK 2 Division of Pathology, European Institute of Oncology and University of Milan, School of Medicine, London, UK Correspondence to: Luca Magnani, e-mail: l.magnani@imperial.ac.uk Keywords: DMXL2, breast cancer, Notch, endocrine therapy, EMT Received: March 23, 2015 Accepted: May 22, 2015 Published: June 04, 2015 ABSTRACT The acquisition of endocrine therapy resistance in estrogen receptor α (ERα) breast cancer patients represents a major clinical problem. Notch signalling has been extensively linked to breast cancer especially in patients who fail to respond to endocrine therapy. Following activation, Notch intracellular domain is released and enters the nucleus where activates transcription of target genes. The numerous steps that cascade after activation of the receptor complicate using Notch as biomarker. Hence, this warrants the development of reliable indicators of Notch activity. DMXL2 is a novel regulator of Notch signalling not yet investigated in breast cancer. Here, we demonstrate that DMXL2 is overexpressed in a subset of endocrine therapy resistant breast cancer cell lines where it promotes epithelial to mesenchymal transition through hyper-activation of Notch signalling via V-ATPase dependent acidification. Following DMXL2 depletion or treatment with Bafilomycin A1, both EMT targets and Notch signalling pathway significantly decrease. We show for the first time that DMXL2 protein levels are significantly increased in ERα positive breast cancer patients that progress after endocrine therapy. Finally, we demonstrate that DMXL2 is a transmembrane protein with a potential extra-cellular domain. These findings identify DMXL2 as a novel, functional biomarker for ERα positive breast cancer.

// Luca Magnani 1 , Darren K. Patten 1 , Van T.M. Nguyen 1 , Sung-Pil Hong 1 , Jennifer H. Steel 1 , Naina Patel 1 , Ylenia Lombardo 1 , Monica Faronato 1 , Ana R. Gomes 1 , Laura Woodley 1 , Karen Page 2 , David Guttery 2 , Lindsay Primrose 2 , Fernandez Garcia 2 , Jacqui Shaw 2 , Patrizia Viola 3 , Andrew Green 4 , Christopher Nolan 4 , Ian O. Ellis 4 , Emad A. Rakha 4 , Sami Shousha 1 , Eric W.-F. Lam 1 , Balazs Győrffy 5 , Mathieu Lupien 6, 7 , R. Charles Coombes 1 1 Department of Surgery and Cancer, Imperial College London, London, UK 2 Department of Cancer Studies, University of Leicester, Leicester, UK 3 Laboratory of Medicine, Histopathology Department, Royal Brompton Hospital, London, UK 4 Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Budapest, HU 5 MTA TTK Lendulet Cancer Biomarker Research Group; 2nd Department of Pediatrics, Semmelweis University and MTA-SE Pediatrics and Nephrology Research Group, Budapest, HU 6 The Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada 7 Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada Correspondence to: Luca Magnani, e-mail: l.magnani@imperial.ac.uk Keywords: breast cancer, estrogen receptor, drug resistance, metastasis, pioneer factors Received: February 18, 2015 Accepted: June 02, 2015 Published: June 15, 2015 ABSTRACT Over 30% of ERα breast cancer patients develop relapses and progress to metastatic disease despite treatment with endocrine therapies. The pioneer factor PBX1 translates epigenetic cues and mediates estrogen induced ERα binding. Here we demonstrate that PBX1 plays a central role in regulating the ERα transcriptional response to epidermal growth factor (EGF) signaling. PBX1 regulates a subset of EGF-ERα genes highly expressed in aggressive breast tumours. Retrospective stratification of luminal patients using PBX1 protein levels in primary cancer further demonstrates that elevated PBX1 protein levels correlate with earlier metastatic progression. In agreement, PBX1 protein levels are significantly upregulated during metastatic progression in ERα-positive breast cancer patients. Finally we reveal that PBX1 upregulation in aggressive tumours is partly mediated by genomic amplification of the PBX1 locus. Correspondingly, ERα-positive breast cancer patients carrying PBX1 amplification are characterized by poor survival. Notably, we demonstrate that PBX1 amplification can be identified in tumor derived-circulating free DNA of ERα-positive metastatic patients. Metastatic patients with PBX1 amplification are also characterized by shorter relapse-free survival. Our data identifies PBX1 amplification as a functional hallmark of aggressive ERα-positive breast cancers. Mechanistically, PBX1 amplification impinges on several critical pathways associated with aggressive ERα-positive breast cancer.