Gamma secretase and Notch as therapeutic targets in cancer.
The complexity in the management of cancers lies in the heterogeneity of the disease which refutes the 'one-size-fits-all' therapeutic approach, but rather calls for tailored and targeted therapies. The other obstacle is the ability of cancer cells to make redundant the pathways that have been inhibited by therapy, and activate alternative routes for survival.
Gamma secretase (GS) is a membrane-embedded multi-protein enzyme composed of presenilin/nicastrin/Aph 1 and Pen 2 . It 'premiered' as a therapeutic target upon the discovery that it cleaves the amyloid precursor protein generating a pathogenic pp-amyloid peptide, which aggregates to form the cerebral plaques underlying Alzheimer's disease .
GS facilitates a unique signalling axis that starts at the cell membrane and culminates in the nucleus. Nicastrin acts as a substrate recognition component of GS at the membrane . Presenilin proceeds to cleave the substrate at the membrane-cytoplasmic interface, liberating its intracellular domain which translocates to the nucleus to regulate transcription of target genes. The result is control and modulation of crucial cellular processes including proliferation, differentiation, migration/invasion and apoptosis. Over the years, more than 30 substrates have been identified to be cleaved by GS (i.e. Notch receptors, CD44, Her4, E-cadherin, EpCAM, MUC-1, etc.) . Amongst them, Notch receptors are the most widely studied for their contribution to development and progression of cancers, and resistance to existing therapies .
The oncogenic tune of the Notch family
The Notch signalling pathway is referred to as the 'aristocracy' on the 'social ladder' of pathways due to its preservation amongst the species and importance in both embryonal development and adult function . Moreover, Notch signalling is found in a rare population of cancer stem cells that are proving to be a challenging target in oncology. Cancer stem cells are thought to lie at the root of malignant disease, and possess properties of self-renewal that allow them to escape the effects of anti-cancer drugs and may serve as the origin of disease relapse .
Preclinical and clinical evidence supports the role of Notch signalling in both haematological malignancies and solid tumours. Over 50% of T-ALL bear activating translocation in the Notch1 receptor while constitutively active Notch is present in B-CLL [8,9]. Some studies report anti-tumour effects achieved by inhibiting Notch in Hodgkin lymphoma and multiple myeloma [10,11]. Notch3 overexpression due to gene amplification is documented in non-small-cell lung cancer and ovarian cancer [12,13]. In the latter, high Notch3 levels are observed in >60% of squamous-cell carcinomas and correlate with advanced stage of disease, resistance to chemotherapy and worse overall survival . High Notch1 levels confer a worse prognosis in head and neck cancer . Signalling via Notch1 seems to contribute to the development of colon cancer through promoting angiogenesis. Furthermore, chemotherapy-based regimens activate Notch1 in colon cancer as a means of developing resistance to treatment . Pancreatic cancer, being one of the most aggressive malignancies and certainly the one with the narrowest therapeutic spectrum, displays dependence on activated Notch1 and Notch3 for proliferation in vitro and in animal models. In addition, concomitant treatment of pancreatic cancer with Notch inhibitors and gemcitabine enhances response to chemotherapy . Glioblastoma multiforme is an aggressive brain tumour where Notch2 expression is recorded, as well as response to its inhibition as judged by reduction in tumour mass and enhanced response to both radiation and chemotherapy .
In breast cancer, high levels of Notch1 mRNA confer worse overall survival . Interestingly, Notchl membrane localisation is noted predominantly in oestrogen receptor-positive (ER-positive) tumours while cytoplasmic expression correlates with lymph node metastasis. High Notch4 levels at the membrane and in the cytoplasm relate to a high proliferative tumour index as determined by Ki67 . Overexpression of Notch1 and Notch4 coincide with the loss of Notch-negative regulator Numb in 50% of breast cancers . In a population of breast cancer stem cells, defined by the [CD44.sup.+]/[CD24.sup.-]/[ESA.sup.+] phenotype, Notch4 activity was dramatically increased (8-fold), while Notch1 exhibited a 4-fold upregulation, as compared with differentiated breast cancer cells pertaining to the same tumour . This indicates that inhibition of Notch in breast cancer may serve to eliminate both the tumour and its stem.
Oestrogen receptor, HER2, EGFR and Notch: communication of signalling pathways
The network of cellular signalling exists to maintain homeostasis of cellular functions and balance tumour-suppressor versus oncogenic signals in favour of the former, to prevent cancer development. Conversely, in a fully transformed malignant cell, these communications serve to ensure that once one pro-survival pathway has been inhibited by therapy, an alternative one is switched on to compensate.
ER-positive breast cancers benefit from well established treatment with tamoxifen . However, during the course of treatment 50% of patients develop resistance and relapse. Notch signalling is activated in breast cancers demonstrating resistance to anti-hormonal agents and promotes growth of ER-negative cells . The mechanism behind this is the ability of Notch to activate ER-related genes in both an oestrogen-dependent and an oestrogen- independent manner. Notch achieves this through the IKK-[alpha] transcription factor-dependent chromatin recruitment of the CSL/MML1/Notch-ICD transcriptional complex to the close proximity of EREs (oestrogen response elements) .
HER2 overexpressing/amplified breast cancers are candidates for targeted therapy with trastuzumab (an anti-HER2 monoclonal antibody), where de novo or acquired resistance is a major hurdle. Activation of Notch signalling serves as one of the mechanisms of resistance to anti-HER2 therapies. Furthermore, treatment of breast cancer with a gamma-secretase inhibitor (GSI) in a murine model resensitises cells to trastuzumab . In colon cancer, treatment with several chemotherapeutic agents (irinotecan, 5-FU and oxaliplatin) induces Notch1-ICD overexpression by elevating enzymic activity of GS through enhanced expression of presenilin and nicastrin. GSI, in this model, serves to resensitise cells to chemotherapy . Crosstalk between EGFR and Notch has been identified in basal-like breast cancer, lung and skin cancers, and glioma . Namely, Notch activation can act to sustain Ras and PI3K/Akt and mTOR signalling [17,27] in the presence of either EGFR or HER2 inhibition, thereby allowing cell survival and proliferation. These studies suggest that activation of Notch may be a means of resistance to both unselective (chemotherapy) and targeted therapies in multiple cancer types, and suggest benefit from combinational therapies that would incorporate GSIs or Notch inhibitors into existing protocols.
To respond or not to respond: signatures will tell
Molecular and gene signatures are novel tools that are used in the era of tailored patient treatment to determine the exact characteristics of the cancer and derive not only prognostic conclusions but also predict response to certain types of treatment. Some of these assays are already incorporated into clinical practice (Oncotype DX, MammaPrint) [28,29].
A gamma-secretase inhibitor MRK-003 was used to determine a gene-response signature in a HER2-driven breast tumour model. The investigation revealed that responding tumours had elevated Notch activity, IL-10 and chemokine signalling . Another similar study used 15 T-ALL cell lines to determine baseline activity of Notch and correlate it to the sensitivity to a GSI. The composite expression score, named the 10 Notch target score (HES-1, HES-4, HES-5, HEY-L, HEY-2, DTX1, C-MYC, NRARP, PTCRA, and SHQ1) was established, which predicts sensitivity to GSIs . It remains to be tested whether this 10-gene-set signature translates to predict sensitivity to GSIs in other tumour types. Another attempt to establish a robust, reproducible and reliable prognostic assay that would indicate tumour sensitivity to GSIs is development of an ex vivo GS activity assay (exo-cell assay) . The method entails processing an excised tumour tissue to obtain a concentrated cell membrane preparation which is then incubated with a labelled GS substrate, which emits a fluorescent signal upon cleavage by the active enzyme. The level of fluorescence corresponds to the degree of cleavage, i.e. to the level of GS activity . Some of these methodologies, alone or in combination, once further optimised and validated, may constitute a clinically approved GSI/Notch inhibition sensitivity test.
Clinical trials with GSIs
Several orally bio-available gamma-secretase inhibitors are being tested in Phase I and II clinical trials that have both proven their clinical efficacy and also revealed the side effects. Due to the unselective nature of the GSIs, simultaneous inhibition of all Notch receptors in the colon gives rise to goblet-cell metaplasia, resulting in diarrhoea. If presented as grade 3/4, this adverse event is taken as the dose- limiting toxicity. Other adverse events recorded have been nausea, fatigue, emesis, pruritus and rash. In general, trials report that the compounds are well tolerated and either stable disease or anti-tumour effects are observed in patients with advanced solid tumours and leukaemias [33,34].
All for one, one for all: collective versus selective targeting of Notch receptors
Due to the complexity of the Notch signalling that leads up to the cleavage by gamma secretase, it is proposed that Notch inhibition can be achieved on multiple levels. Starting at the top of the membrane-to-nucleus axis, the Notch cascade of events can be blocked using several approaches. These include the Notch ligand-blocking antibodies . Ligands such as
Jagged and Delta interact with the Notch receptor on the cell surface to initiate the conformational change in the Notch extracellular domain . This conformational change can be prevented with the blocking antibodies against the Notch negative regulatory domain . Next, the ADAM protease inhibitors can be used to prevent the sequential alpha cleavage that occurs in the Notch extracellular domain and renders it 'recognisable' for the gamma secretase . The commonality between these approaches is collective inhibition of all Notch receptors that are expressed and signal in the given context. Given the severity of side effects that result from the simultaneous inhibition of Notch receptors, particularly Notch1 and Notch2, development of therapeutic monoclonal blocking antibodies against each individual Notch is under way . This will allow for a more specific treatment approach, reduced side effects and elucidation of discrete functions of individual Notch receptors.
Targeting nicastrin: two birds with one stone
Last but not least, the gamma-secretase complex harbours another attractive and emerging therapeutic target. Nicastrin is deemed the gatekeeper of the enzyme . It is also the sole GS component with a single-pass transmembrane domain and a large extracellular segment  that is potentially targetable with a blocking antibody. During GS composition, nicastrin represents a structural scaffold for other member proteins to assemble on, while within a functional complex, it executes a substrate recognition role [3,38]. Nicastrin overexpression has proven sufficient to induce GS activity without altering the levels of the catalytic subunit presenilin . Inhibition of nicastrin in breast cancer cells by gene silencing disrupts complex formation, reduces GS activity and activation of Notch receptors. Nicastrin shRNA induces Notch inhibition in basal-like breast cancer cells, sensitising them to anti-proliferative effects of EGFR inhibition . Furthermore, nicastrin protein levels are increased in response to oxaliplatin treatment in colon cancer, inducing Notch signalling as a mechanism of resistance to these chemo-agents. Nicastrin is thought to possess a GS-independent function as well, particularly within the p53 signalling pathway and regulation of cell death . Nicastrin displays differential expression between normal and cancer tissue in several organs. Low levels of nicastrin are noted in normal breast, thyroid, lung and colon tissue, while the counterpart malignant tumours of these organs display high nicastrin levels. In breast cancer, high nicastrin levels confer worse overall survival in the ET-negative patients, whose tumours are by nature deemed more aggressive and deprived of anti-hormonal therapy as an option of treatment. Preclinical testing of nicastrin-blocking antibodies has shown that they exert substantial anti-proliferative and anti-invasive effects in breast cancer cells . Further studies are under way to elucidate the nicastrin propriety versus nicastrin role within gamma secretase, as are the characterisation studies of the anti-nicastrin monoclonal antibodies for the treatment of invasive breast cancer.
GS enzyme and Notch receptors bear substantial oncogenic potential and contribute to the development and progression of malignant disease and resistance to multiple treatment regimens. Promising preclinical and clinical data announce GS inhibitors, as well as Notch and nicastrin-blocking antibodies, as powerful anti-cancer agents that may complement the existing arsenal of anti-cancer drugs in the future.
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Aleksandra Filipovic, Georgios Giamas, R Charles Coombes and Justin Stebbing
Department of Surgery and Cancer, Division of Cancer, Imperial College London,
Correspondence to: Aleksandra Filipovic
School of Medicine
Imperial College London
MRC Cyclotron Building
Hammersmith Hospital Campus
Du Cane Road, London W12 0NN
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|Title Annotation:||Feature Article|
|Author:||Filipovic, Aleksandra; Giamas, Georgios; Coombes, R. Charles; Stebbing, Justin|
|Publication:||Advances in Breast Cancer|
|Date:||Sep 1, 2010|
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