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The Notch-3 receptor: A molecular switch to tumorigenesis?
Accepted Manuscript Tumour Review The Notch-3 receptor: a molecular switch to tumorigenesis? Shakeel Inder, Sinead O'Rourke, Niamh McDermott, Rustom Manecksha, Stephen Finn, Thomas Lynch, Laure Marignol PII: DOI: Reference:
S0305-7372(17)30143-3 http://dx.doi.org/10.1016/j.ctrv.2017.08.011 YCTRV 1669
To appear in:
Cancer Treatment Reviews Cancer Treatment Reviews
Received Date: Revised Date: Accepted Date:
23 May 2017 25 August 2017 26 August 2017
Please cite this article as: Inder, S., O'Rourke, S., McDermott, N., Manecksha, R., Finn, S., Lynch, T., Marignol, L., The Notch-3 receptor: a molecular switch to tumorigenesis?, Cancer Treatment Reviews Cancer Treatment Reviews (2017), doi: http://dx.doi.org/10.1016/j.ctrv.2017.08.011
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The Notch-3 receptor: a molecular switch to tumorigenesis? Shakeel Inder,1,2 Sinead O’Rourke,1 Niamh McDermott,1 Rustom Manecksha ,
2
Stephen Finn,
3
Thomas Lynch,2 Laure Marignol1 1
Translational Radiobiology and Molecular Oncology, Applied Radiation Therapy Trinity, Trinity College Dublin, Dublin, Ireland 2 Department of Urology, St James’s Hospital, Dublin 3 Department of Histopathology. St James’s Hospital, Dublin
Corresponding Author Laure Marignol, PhD Translational Radiobiology and Molecular Oncology Applied Radiation Therapy Trinity Trinity Centre for Health Sciences St James’s Hospital Dublin 8 Ireland Tel:+353 1 896 3255 Email: [email protected] Key words: Notch, cancer, biomarkers
1
Abstract The Notch pathway is a highly conserved pathway increasingly implicated with the progression of human cancers. Of the four existing receptors associated with the pathway, the deregulation in the expression of the Notch-3 receptor is associated with more aggressive disease and poor prognosis. Selective targeting of this receptor has the potential to enhance current anti-cancer treatments. Molecular profiling strategies are increasingly incorporated into clinical decision making. This review aims evaluate the clinical potential of Notch-3 within this new era of personalised medicine.
Introduction The Notch pathway is an evolutionarily conserved signalling system that regulates cell proliferation, differentiation, cell fate determination and stem/progenitor cell self-renewal in both embryonic and adult organs [1, 2];but is also essential to the regulation of blood vessel structure [3, 4]. The Notch pathway is activated by the interaction between its ligands (Jagged 1 & 2, and Delta-like homologues 1-4) and receptors (Notch-1 to 4) which induces cleavage of the Notch receptors and culminates in the release of the Notch Intracellular domain (NICD). Nuclear translocation of the NICD and binding to the transcriptional repressor RBPJ (recombining binding protein suppressor of hairless) enables the induction of the expression of downstream target genes, such as HES-1 (Figure 1) [5, 6]. This process may be negatively regulated by the NUMB protein, an important cell fate determinant [7, 8].
The role of Notch signalling in malignancy was first reported in 1991 when the previously uncharacterized locus in a t(7;9)(q34;q34.3) chromosomal translocation from a case of human T lymphoblastic leukemia (T-ALL) contained a gene highly homologous to the Drosophila gene NOTCH, later named NOTCH-1 [9]. This damage results in the presence of a truncated NOTCH-1 protein with the ability to drive oncogenic transformation in rat kidney cells in vitro [10]. When the truncated protein was detected in over 65% of all T-ALL patients [11], the Notch pathway emerged as a key driver of T-ALL development. Since then, the pathway has been attributed both oncogenic and tumour suppressive properties [12], and associated with altered microvasculature and vascular function within tumours [13, 14].
The Notch-3 receptor was first identified in the neuroepithelium [15]. Mutation of the NOTCH-3 gene is most commonly associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoecephelopathy (CADASIL)- a degenerative vascular disease that increases patients’ risk of stroke [16]. But elevated expression of this receptor is increasingly documented in cancers including gastric, pancreatic, colorectal, hepatocellular, lung, cervical, ovarian and prostate [17]. 2
Higher Notch-3 expression has been linked to rapid malignant progression, abnormal differentiation, metastasis and poorer prognosis (Figure 1) [17].
The known interaction of Notch signalling with other major signalling pathways [18] such as Wnt and TFGß, further strengthen the potential of this pathway for the development of novel anti-cancer treatment approaches [19, 20]. Activation of Wnt signalling with Lithium-Chloride (LiCl) in non-small cell lung cancer (NSLCs) cell lines was associated with a significant increase in NOTCH-3 expression coupled with an increase in HES-1 mRNA levels, when compared to untreated controls [21]. In these same cells, the inhibition of NOTCH-3 by small interfering RNA decreased invasion ability and reduced that induction of interleukin (IL-6) by transforming growth factor (TGF) in vitro [22].
The clinical potential of molecular profiling in treatment decision making is increasingly recognised, [23, 24] but the development of these personalised approaches remains limited by the paucity of adequate biological markers of disease with prognostic and therapeutic value. The implication of the Notch-3 receptor in both the development of an abnormal vasculature and the emergence of a malignant phenotype highlights this receptor as potential key to tumorigenesis. This review aims to evaluate the potential for the Notch-3 receptor for the development of novel personalised medicine strategies.
Notch-3 receptor: activation and structure The structure of Notch receptors is essential for Notch activity in mammals [25]. Notch receptors are transmembrane proteins, with a heterodimeric structure consisting of the Notch extracellular domain (NECD), a single transmembrane domain, and the Notch intracellular domain (NICD) (Figure 1) [26]. The NECD of the receptors in mammals contains approximately 26-36 epidermal growth factor (EGF)-like domains, and three cysteine rich repeats, known as Lin repeats. These domains enable ligand-receptor interaction. For instance, the interaction of delta-like ligands of a neighbouring cell with EGF 11 and 12 of the extracellular domain activates the Notch signalling pathway [27] Ligand binding induces cleavage mediated by the enzyme complex ℽ-secretase [18]. Following proteolytic cleavage, further activation of Notch signalling is mediated by the NICD, whose nuclear translocation changes recombining binding protein suppressor of hairless (RBPJ) proteins to transcription activators. Together with Master mind-like polypeptides, this Notch transcription activation complex regulates downstream gene expression [28]. While the activation mechanisms of the Notch pathway appear well described, the distinct functions of each receptor remain poorly understood. Studies attempting to compare the structure and 3
function of the Notch receptors progressively highlight distinctive features of the Notch-3 receptor that may explain its emerging key role in tumorigenesis. For instance, although the HES-1 transcription factor is the most commonly known Notch target (Figure 1) [29, 30], the Notch-3 receptor appears a poor activator of specific HES-1 target genes. Transfection of the Notch-3 NICD in the human chorion carcinoma JEG cell line failed to trigger HES-1 promoter activity irrespective of the concentration of RBP-Jk available, whereas Notch-1 NICD overexpression yielded strong activation of the promoter [31, 32]. Further evidence suggest that Notch-3 and Notch-4 diverge from the known structure of the Notch-1 and Notch-2 receptors [33]. All Notch receptors carry a PEST sequence near the C-terminus domain involved in negative regulation of the pathway and the degradation of the NICD in the nucleus [32]. This sequence is located beside the Notch transcriptional activation domain (TAD), which is capable of autonomous transcriptional activation [34]. The TAD shows substantial evolutionary divergence among the four mammalian Notch receptors. But unlike other Notch receptors (Notch-1, Notch-2), Notch-3 contains both TAD and cytokine response regions (NCR) post cleavage [35, 36]. This particularity could explain the ability of Notch-3 to repress HES-1 signalling [37, 38] A frameshift mutation in the Notch-3 receptor was identified at amino acid position 1802 [39] and upstream the PEST domain of the receptor in triple negative breast cancer [40]. Mutations in Notch receptor genes are common and were present in the majority of the 905 tumour lines examined by Mutvei et al., with 5% of cell lines carrying more than one mutation [39]. Frameshift mutations were the second most commonly observed mutations in Notch receptors [39]. In this extensive dataset, the type and frequency of these mutations varied according to tumour types. Cell lines derived from tumours of the prostate and endometrial cells presented the highest frequency in Notch mutations. The oesophagus and urinary tract contained considerable less mutations but still at a significant frequency of greater than 25% [39]. The mutation frequency of Notch was determined to fall below that of Ras, but within a similar range to the ErbB1-4 and APC genes [39]. The range of frequencies when examining the different receptors and ligands of the Notch pathway varied from 7.3% in Notch-1, down to 2.9% in Delta-like ligand 4. [39] The significance of this unique Notch-3 mutation is yet to be elucidated [39] but is known to result in the loss of the functioning PEST domain at the Cterminus end of the protein [39, 40]. PEST is involved in the tight regulation of the ICD domain and deletion increases stability and half-life of the NICD [41]. This may explain over expression of the signalling pathway in cancer cells [41].
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Notch-3 receptor: a driver of the malignant phenotype The clinical evidence for the role of Notch-3 overexpression when compared to benign tissue as a driver of malignancy is rapidly accumulating across a variety of cancer sites. In the cervix, immunochemistry analysis of 168 biopsy specimens identified increasing NOTCH-3 staining intensity with increasing disease stage: 86.7% (26/30) of pre-cancer and 85.7% (84/98) of analysed tumour tissues showed significant cytoplasmic and nuclear NOTCH-3 positivity. In pre-cancerous lesions, the presence of high cytoplasmic NOTCH-3 staining was dominant in the 87.5% of specimens testing positive for human papilloma virus (HPV)-16 [42]. In breast cancer, in situ mRNA hybridisation identified high NOTCH-3 expression in 6 of the 50 tumours profiled by Reedijk et al. [43], which correlated with increased predicted mortality at 10 years. In this small sample, these tumours were almost exclusively triple negative (negative for ErbB2, and both the estrogen and progesterone receptors expression). In inflammatory breast cancer patients, elevated nuclear expression of NOTCH-3 was detected in 80% of the 25 cases tested [44]. More recently mechanistic studies using antagonist and agonist Notch-3 antibodies demonstrated that Notch-3 activation promotes the development of basal breast cancer [45].
In lung cancer, NOTCH-3 immunostaining intensity was highest in non-small cell (NSCLC) patient biopsy specimens (adenocarcinoma (n=24); squamous cell carcinoma (n=20)) and lowest small cell carcinoma (n=13) when compared to neighbouring normal tissue [46]. This result was confirmed by a meta-analysis of 19 studies involving 3663 NSCLC patients [47]. This work further identified that NOTCH-3 expression in NSCLC tissues significantly correlates with the overall survival rate of NSCLC patients. The overexpression of both Notch-3 mRNA and immunostaining levels was reported in tongue squamous cell carcinoma patient specimens. Expression was elevated in the majority of the 74 specimens profiled, but failed to correlate with clinical staging [48, 49]. Fibroblasts in the cancer stroma, especially those adjacent to the tumour periphery, showed positive staining for NOTCH-3 in 31 out of 93 cases examined by immunochemistry [50]. In ovarian cancer, high mRNA expression level of Notch-3 was associated with better overall survival for all cancers but the serous and endometrioid subtypes [51, 52]. NOTCH-3 mRNA levels and immunostaining intensity were elevated in intestinal-type carcinomas [53] Elevated NOTCH-3 NICD expression levels were elevated in tumour-bearing bones compared to control bones in a murine model of multiple myeloma [54]. In gallbladder malignancy, increased NOTCH-3 immunostaining intensity was associated with larger tumour size, invasion, metastasis and low curability rates in both
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squamous cell/adenosquamous carcinomas (n=46) and adenocarcinomas (n=80) [17]. Zhang et al. identified elevated NOTCH-3 mRNA levels in over 71.8% of the 32 cases of hepatocellular carcinomas studied which positively correlated with alpha-fetoprotein but negatively correlated with both differentiation grade and survival [55]. In glioblastoma, NOTCH-3 was moderately upregulated in 40 % of the 15 samples tested [56]. In these disease, NOTCH-3 amplification was associated with significantly worse outcome (median survival 10 vs. 28 months, Log-Rank test ) compared to tumours with non-amplified locus in the 60 tumour biopsies analysed. The amplification resulted in a significant elevation of NOTCH-3 protein levels, when compared to samples with non-amplified locus [57] . An increase in NOTCH-3 expression was also reported in melanoma specimens, possibly enabling differentiation between malignant melanoma and benign Nevi [58]. In a study carried out by Man et al., NOTCH-3 was expressed in 92.5% of patients with Epstein-Barr Virus (EBV) positive tumours and all other nasopharyngeal tumours, demonstrating that Notch-3 was heavily implicated in the development of nasopharyngeal cancer [59]. But conflicting data have been reported in regards to the implication of Notch-3 in renal malignancies [60]. In a cohort of 80 patients analysed by tissue microarray, the presence of NOTCH-3 was better correlated with renal carcinoma when the latter was present in the right kidney [60]. Further analysis of 84 cases detected low level positivity for NOTCH-3 staining in renal tumours staged T1 or T2 but the results were not statistically significant [61]. Finally, the elevation of Notch-3 mRNA and immunostaining intensity was documented in prostate cancer. Although one study reported that NOTCH-3 was not expressed in the metastatic prostate cancer cell lines LNCaP, PC-3 and PC-3M) [62], another identified high expression of both HES-6 and NOTCH-3 in VCaP and LnCaP96 cells and proposed that these two markers could identify aggressive prostate cancers [63]. Further reports supporting the involvement of Notch-3 in prostate tumorigenesis exist. Notch-3 has a critical role to play in the development of the prostate gland as well as prostate cancer [64]. NOTCH-3 expression was positively correlated with the grading of prostate cancer [65, 66]. Furthermore, high levels of NOTCH-3 post-radical prostatectomy were indicative of recurrence [67].
A reduction in Notch-3 expression can however also be indicative of malignancy. In lung cancer specimens, NOTCH-3 expression in the cancer tissue was weaker than that of the corresponding non-tumor tissue in the SCLC group [46]. This also appears the case in thyroid carcinoma. Medullary thyroid carcinomas patient specimens analysed were deficient in NOTCH-3 intracellular domain 6
while high levels were detected in normal thyroid epithelial cells [68]. Analysis of a tissue microarray of normal and pathologic thyroid biopsies from 155 patients identified that NOTCH-3 expression levels regressed across decreasingly differentiated, increasingly malignant thyroid specimens and were predictive of survival [69].
Notch-3 receptor: a molecular switch The detailed evaluation of the molecular consequences of Notch-3 activation using siRNA, neutralising antibodies or Notch inhibitors is under scrutiny. As expected, Notch-3 appears directly implicated in proliferation and invasion of cancer cells (Figure 1). In MCF-7 breast cancer cells, increased expression of the Notch-3 N3ICD was associated with arrest of cell cycle at the G0/G1 phase [70], but in Erb B2-Negative tumour cell lines down regulation of Notch-3 lead to decreased proliferation and increased apoptosis [71]. As a result Notch-3 targeted therapies may be of most benefit for the treatment of Erb B2-negative breast tumours [71, 72]. Activation of Notch-3 was additionally associated with the upregulation of IL-6 in breast cancer cells [73]. IL-6 has already been established as a major mediator of inflammatory responses in humans [74], and a critical player within the pathophysiology of cancer. [73].
In NSCLC, manipulation of Notch-3 expression lead to a modification in the expression levels of NOTCH-1, HES-1 and JAGGED-1 [46], and suggestions of its regulation by Protein Kinase Ci [75]. Characterisation of the impact of Notch-3 manipulation on cell adhesion, motility and EpithelialMesenchymal Transition in vitro concluded that Notch-3 could act as a tumour suppressor in NSCLC but as a tumour promoter in SCLC [46]. Notch-3 inhibition using siRNAs prevented cell proliferation in NSCLC cell lines. In SCLC, Notch-3 inhibition stimulated cell proliferation [46, 76]. Similarly, treatment of follicular thyroid cancer cell lines with the histone deacetylase inhibitor AB3 increased NOTCH-3 NICD levels, decreased the expression of neuroendocrine tumour markers (ASCL 1 and CgA) and affected cell proliferation [68]. In vivo, NOTCH-3 NICD was essential to the reduction of tumour burden through apoptosis [69]. In prostate cancer cells, the regulation of NOTCH-3 by p38 MAPK, including its mRNA half-life time, was key to the induction of luminal differentiation [77]. In T-ALL, the tumorigenic properties of Notch-3 may be mediated through the regulation of miR-223 and the downstream inhibition of the tumour suppressor FBXW7. This mechanism may be key to the response of T-ALL to gamma-secretase inhibitor (GSI) treatment, as miR-223 inhibition prevented TALL resistance to these agents [78]
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The biological impact of Notch-3 deregulation is associated with the induction of chemoresistance (Figure 1). Patients with advanced NSCLC whose tumours overexpressed NOTCH-3 were at a higher risk of resistance and poor overall survival following platinum-based chemotherapy [79]. These tumour specimen also overexpressed the cancer stem cell markers ALDH1A1 and CD44. Blockade of Notch-3 using siRNAs in A459 lung cancer cells lead to a reduction in LC3II expression and prevented autophagy activation [79, 80]. In nasopharyngeal cell lines, increased sensitivity to cisplatin treatment following the suppression of Notch-3 by siRNAs was also demonstrated [59]. In breast cancer cells, high NOTCH-3 expression correlated with reduced chemoresistance [81], due to the negative regulation of the drug efflux pump P-glycoprotein by NOTCH-3. The study additionally proposed the epigenetic silencing of NOTCH-3 through methylation as an underlying mechanisms of chemoresistance in breast cancer [81]. In triple negative breast cancer patients, high coverage whole genome sequencing together with transcriptome and whole exome sequencing identified that structural variations are associated with the amplification of NOTCH-3. But the receptor appears particularly important in the chemoresponse of ovarian cancer cells [82, 83]. Chemoresistant ovarian cancers were associated with a higher level of NOTCH-3 than NOTCH-1 [83]. In vitro, silencing of NOTCH-3 lead to improved sensitivity of ovarian cancer cells to paclitaxel [83]. The induction of miR136 in ovarian cancer cells repressed Notch-3 mediated signalling and re-sensitized paclitaxelresistant ovarian cancer cells, likely through a reduction in cancer stem cells (CSC) activities [84]. The Notch pathway is well documented to promote the growth of CSCs and thereby contribute to the resistance of tumours to chemotherapy and radiotherapy [85]. In breast cancer, often referred to as a “stem cell disease”, Notch-3 and its associated active pathway has been attributed a role in the self-renewal of CSCs, and the promotion of cell survival under hypoxic conditions [73]. In hepatoma cell lines, down regulation of NOTCH-3 affected sensitivity to cisplatin in a mechanisms proposed to involve the interaction of Notch-3 with the Wnt/beta-catenin pathway to regulate stemness [55]. Similarly, the downregulation of NOTCH-3 using lentilvirus in melanoma cell lines resulted in a reduction in the expression levels of stem cell markers, such as CD113 and CD271, and severely compromised the tumorigenicity of the cells in vivo. Taken together this clear association between Notch-3 expression and the CSC population strengthen the need to evaluate Notch-3 targeted therapeutic approaches. These may be of particular relevance to prevent undesirable effects of other molecular approaches. Indeed, treatment of EGFR-mutated lung cancer cell lines with the EGFR tyrosine kinase inhibitor erlotinib was associated with an enrichment of the ALDH(+) stem-like cell population through EGFR-dependent activation of Notch-3, likely compromising treatment efficacy [86]. Similarly, the inhibition of NOTCH-2 by gliotoxin in chronic lymphocytic leukaemia (CLL) cells was associated with an increase in NOTCH-3 expression [87]. 8
Notch-3 receptor: a driver of vascular expansion Components of the Notch system were reported to be expressed at different levels during vascular development and therefore, its role in vascular expansion during tumorigenesis is likely. The angiogenetic properties of Notch-3 has been recently investigated by Liu H et al. The authors described Notch-3 as a critical regulator of blood vessels formation in both a developmental phase and pathological event [88]. In triple negative breast cancer, microvascular density correlated with elevated Notch-3 mRNA and protein levels, but not those of the vascular endothelial growth factor, suggesting Notch-3 as a potentially key therapeutic target of neovascularization [89]. In liver cancer, immunohistochemical analyses of patient specimens revealed that only the increased level of NOTCH-3 was positively correlated with vascular invasion [90]
Notch-3 stimulates the growth of vascular smooth muscle cells via inhibition of a cycline dependent kinase [91]. The receptor is highly expressed in the response to vascular damage [91], possibly through the action of miR- 206 [92]. The exact function of Notch-3 in relation to these vascular events remains poorly characterised [93], but a role for Notch-3 in lung hypertension is emerging [94]. Aberrant Notch signalling was noted within multiple other pathological processes, including the degenerative disease CADASIL [16], Alagille syndrome and spondylocostaldysostosis [95].
The role of Notch-3 in the vasculature highlights the potential key relevance of this receptor to the cellular adaptation of cancer cells to hypoxia. We have proposed this link within the context of prostate cancer [96] and demonstrated, like others [97, 98], that Notch-3 expression may be elevated in response to hypoxic exposure and that hypoxic conditions may alter the activation of Notch-3 mediated signaling [99]. In breast cancer cells, Notch-3 activity was essential to the induction of carbonic anhydrase 9 (CA9) under hypoxic conditions [100]. The interaction of CA9 with miR-34a, which acts as a feedback loop to the modification of NOTCH-3 levels, was proposed to participate in the regulation of human mammospheres under these conditions [101].
But the role of Notch-3 could potentially further lie in the regulation of tumour dormancy (Figure 1) [102]. In T-ALL and colorectal cancer cells, escape from dormancy was associated with an increased in the expression of the Notch ligand DLL4 and the Notch-3 receptor [103]. The interaction between DLL4 and Notch-3 may repredent a key molecular switch to the promotion of tumorigenicity [104], which leads to the regulation of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1)
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levels [102]. In breast cancer, this mechanisms could drive recurrence following HER2/neu pathway inhibition [105].
Notch-3 targeted therapies The ability to control the intensity of Notch signalling has high therapeutic relevance [106] and a number of therapeutic approaches are currently under development. These include gammasecretase inhibitor, Alpha-secretase inhibitor and immunotherapy with neutralising antibodies (Figure 1) [106-110] C-secretase is a protein complex that is activated by the binding of Notch ligands (Jag 1, Jag 2, and DLL 1-3) to Notch receptors. It was identified in 1999 and the therapeutic potential of its inhibition has been under intense scrutiny in many cancer sites [111]. An increase sensitivity of cancer cells to cytotoxic chemotherapy and radiotherapy can be achieved with the use of gamma secretase inhibitors (GSI). This effect may be in part dependent on the induction of apoptosis by Notch-3 in endothelial cells [112]. In ovarian cancer cell lines, sensitisation to paclitaxel was of the same magnitude following GSI treatment or Notch-3 knockdown [83]. This suggests Notch-3 targeting as a strong approach to inhibit Notch signalling and possibly reduce the known side effects of GSIs. In NSCLC, combination of GSIs with radiation prevented radiation-induced increase in NOTCH-3 expression and increased cellular radio sensitivity [113]. In glioblastoma tumour-initiating cells, 17 genes representative of active Notch signalling components including NOTCH-3, identified in 49.3% of the tumours profiled in The Cancer Genome Atlas expression dataset, discriminated good from poor responder to GSI treatment [114]. This data suggests that pre-treatment screening of Notch activity could identify the subgroup of patients most likely to respond to treatment.
The second class of agents considered for Notch inhibition are monoclonal antibodies [115]. A more recent example of this group of inhibitors is the antibody OMP-59R5,[116] which targets both Notch3 and Notch-2 receptors. The antibody, alone or in combination with chemotherapy, proved efficient in inhibiting the expression of Notch target genes and reducing tumour-initiating cell frequency in a broad spectrum of epithelial tumours, including breast, lung, ovarian, and pancreatic cancers [116]. The antibody which just recently entered phase 1 trials for dosage treatment in patients, was developed to specifically target the Notch-2 receptor and consequently prevent ligand binding. In further studies it was observed that the antibody also binds with Notch-3 and further prevents interactions with this specific receptor and its associated ligands. Those treated with the drug were showed non-linear clearance of tumours present from pancreatic cancer, with diarrhoea reported as the most extreme side effects of the treatment [116]. A similar approach is showing exciting results 10
in NSCLC. In cell line models and patient-derived xenograft tumours, the dual targeting of EGFR and Notch2/3 receptors with the antibody CT16 prevented acquired resistance to EGFR inhibitors and radiation through the reduction of the cancer stem cells compartment [117]. These approaches may be particularly important for the management of tumours with Notch-3 mutations. Bernasconi-Elias P et al [41] identified that mutation of the NRR domain of the Notch-3 receptor in T-ALL cell lines resulted in an increase in ligand-dependent Notch-3 signalling. This mutation is associated with a chemical substitution where a hydrogen bond initially formed between a backbone nitrogen and oxygen side chain is broken. This weakens the interaction between the nitrogen and oxygen side-chain, both which are located close to the S2 site of cleavage in the receptor. This weakened interaction possibly makes the S2 site more accessible by proteases to cleave the NICD domain from the receptor. This hypothesis is supported by the elevated levels of NOTCH-3 NICD in cell T-ALL cell lines [41]. The growth and survival of cancerous cell lines such as TALL proved to be dependent on this specific mutation. Upon the addition of an inhibitory antibody of the Notch-3 receptor in mice, tumour growth was compromised [41]. Pre-treatment determination of Notch-3 mutational status may thus represent a key step in the identification of the subset of patients most likely to benefit from Notch-3 targeted therapy. But other strategies are emerging. Emerin was proposed as a potential target within the Notch pathway [118]. This protein has the ability to down regulate Notch signalling. The inner membrane protein induces retention of the NICD once cleaved from the Notch receptor, preventing entrance into the nucleus to activate target genes. Emerin thus represent a prime therapeutic target to selectively control the activation of Notch signalling [118]. Similarly, the possible interaction of the pathway with survivin, an inhibitor of apoptosis abnormally expressed cancer, represent an attractive strategy to Notch targeting [119].
Conclusion The evidence for the role of the Notch-3 receptor in tumorigenesis is rapidly accumulating. This Notch receptor and the pathway in whole have been specifically targeted in the hopes of developing a successful anti-cancer treatment. There has already been positive results demonstrating significant tumour regression when effectively “switching off” the Notch pathway. Further evaluation of the exact impact of Notch-3 mediated signalling on tumorigenesis is warranted and will likely guide the development of more potent, specific, anti-cancer agents.
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Figure1: Schematic representation of the activation and downstream functions of the Notch-3 receptor in tumorigenesis. (1) The interaction of Notch ligand with the notch extra cellular domain (NECD) of the receptor leads to the cleavage of the receptor by the gamma-secretase enzyme, and the release of the notch intra-cellular domain (NICD). The nuclear translocation of the NICD and its interaction with the transcription factor RBPJ (recombining binding protein suppressor of hairless) enables the induction of the expression of downstream Notch-3 target genes, but not HES-1. (2) Activation of Notch-3 leads to the induction of a variety of biological processes driving tumorigenesis, including angiogenesis and the release from tumour dormancy. (3) The biological functions of Notch-3 signalling participate in chemoresistance in many cancer types, such as ovarian and lung cancer; promote metastasis and a poor patient prognosis (e.g non-small cell lung cancer (NSCLC), ovarian and glioblastoma). (4) The increasingly described role of Notch-3 in tumorigenesis warrants the investigation of Notch-3 inhibition therapeutic strategies. Gamma secretase inhibitors and antibodies targeted against the receptor are already yielding promising results. (+) denote a positive action of Notch-3, (-) a repressive action.
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18
Notch ligands Jagged 1,2, DLL 1-4
Elevated expression in most cancers.
4 NECD
Notch-3
Frameshift mutations.
Gamma secretase
Inhibitors
NICD
Extra-cellular space
Cytoplasm
Cleavage
1
TAD and NCR present
NICD
NICD
RPBJ
-
Nucleus
+
Notch-3 target genes
HES-1
Nucleus P300 / RBPJ
2
Cell migration
Proliferation
Differentiation
Apoptosis
Lung + cancer
Autophagy
P-glycoprotein
Breast + cancer
IL-6
Angiogenesis
+ -
-
SCLC NSCLC
Tumour dormancy
3 Chemoresistance + Ovarian, lung cancer
Metastasis
Poor prognosis
Conflict of interest
All authors have no conflict of interest to declare.
Highlights
Deregulation of Notch-3 is associated with more aggressive, often chemo resistant malignancies
Notch-3 acts as a molecular switch to a variety of biological processes including angiogenesis and the release from tumour dormancy.
Therapeutic strategies against Notch-3 are showing promise in preventing tumour growth alone or in combination with chemo- or radio- therapy.
19
S0305-7372(17)30143-3 http://dx.doi.org/10.1016/j.ctrv.2017.08.011 YCTRV 1669
To appear in:
Cancer Treatment Reviews Cancer Treatment Reviews
Received Date: Revised Date: Accepted Date:
23 May 2017 25 August 2017 26 August 2017
Please cite this article as: Inder, S., O'Rourke, S., McDermott, N., Manecksha, R., Finn, S., Lynch, T., Marignol, L., The Notch-3 receptor: a molecular switch to tumorigenesis?, Cancer Treatment Reviews Cancer Treatment Reviews (2017), doi: http://dx.doi.org/10.1016/j.ctrv.2017.08.011
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The Notch-3 receptor: a molecular switch to tumorigenesis? Shakeel Inder,1,2 Sinead O’Rourke,1 Niamh McDermott,1 Rustom Manecksha ,
2
Stephen Finn,
3
Thomas Lynch,2 Laure Marignol1 1
Translational Radiobiology and Molecular Oncology, Applied Radiation Therapy Trinity, Trinity College Dublin, Dublin, Ireland 2 Department of Urology, St James’s Hospital, Dublin 3 Department of Histopathology. St James’s Hospital, Dublin
Corresponding Author Laure Marignol, PhD Translational Radiobiology and Molecular Oncology Applied Radiation Therapy Trinity Trinity Centre for Health Sciences St James’s Hospital Dublin 8 Ireland Tel:+353 1 896 3255 Email: [email protected] Key words: Notch, cancer, biomarkers
1
Abstract The Notch pathway is a highly conserved pathway increasingly implicated with the progression of human cancers. Of the four existing receptors associated with the pathway, the deregulation in the expression of the Notch-3 receptor is associated with more aggressive disease and poor prognosis. Selective targeting of this receptor has the potential to enhance current anti-cancer treatments. Molecular profiling strategies are increasingly incorporated into clinical decision making. This review aims evaluate the clinical potential of Notch-3 within this new era of personalised medicine.
Introduction The Notch pathway is an evolutionarily conserved signalling system that regulates cell proliferation, differentiation, cell fate determination and stem/progenitor cell self-renewal in both embryonic and adult organs [1, 2];but is also essential to the regulation of blood vessel structure [3, 4]. The Notch pathway is activated by the interaction between its ligands (Jagged 1 & 2, and Delta-like homologues 1-4) and receptors (Notch-1 to 4) which induces cleavage of the Notch receptors and culminates in the release of the Notch Intracellular domain (NICD). Nuclear translocation of the NICD and binding to the transcriptional repressor RBPJ (recombining binding protein suppressor of hairless) enables the induction of the expression of downstream target genes, such as HES-1 (Figure 1) [5, 6]. This process may be negatively regulated by the NUMB protein, an important cell fate determinant [7, 8].
The role of Notch signalling in malignancy was first reported in 1991 when the previously uncharacterized locus in a t(7;9)(q34;q34.3) chromosomal translocation from a case of human T lymphoblastic leukemia (T-ALL) contained a gene highly homologous to the Drosophila gene NOTCH, later named NOTCH-1 [9]. This damage results in the presence of a truncated NOTCH-1 protein with the ability to drive oncogenic transformation in rat kidney cells in vitro [10]. When the truncated protein was detected in over 65% of all T-ALL patients [11], the Notch pathway emerged as a key driver of T-ALL development. Since then, the pathway has been attributed both oncogenic and tumour suppressive properties [12], and associated with altered microvasculature and vascular function within tumours [13, 14].
The Notch-3 receptor was first identified in the neuroepithelium [15]. Mutation of the NOTCH-3 gene is most commonly associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoecephelopathy (CADASIL)- a degenerative vascular disease that increases patients’ risk of stroke [16]. But elevated expression of this receptor is increasingly documented in cancers including gastric, pancreatic, colorectal, hepatocellular, lung, cervical, ovarian and prostate [17]. 2
Higher Notch-3 expression has been linked to rapid malignant progression, abnormal differentiation, metastasis and poorer prognosis (Figure 1) [17].
The known interaction of Notch signalling with other major signalling pathways [18] such as Wnt and TFGß, further strengthen the potential of this pathway for the development of novel anti-cancer treatment approaches [19, 20]. Activation of Wnt signalling with Lithium-Chloride (LiCl) in non-small cell lung cancer (NSLCs) cell lines was associated with a significant increase in NOTCH-3 expression coupled with an increase in HES-1 mRNA levels, when compared to untreated controls [21]. In these same cells, the inhibition of NOTCH-3 by small interfering RNA decreased invasion ability and reduced that induction of interleukin (IL-6) by transforming growth factor (TGF) in vitro [22].
The clinical potential of molecular profiling in treatment decision making is increasingly recognised, [23, 24] but the development of these personalised approaches remains limited by the paucity of adequate biological markers of disease with prognostic and therapeutic value. The implication of the Notch-3 receptor in both the development of an abnormal vasculature and the emergence of a malignant phenotype highlights this receptor as potential key to tumorigenesis. This review aims to evaluate the potential for the Notch-3 receptor for the development of novel personalised medicine strategies.
Notch-3 receptor: activation and structure The structure of Notch receptors is essential for Notch activity in mammals [25]. Notch receptors are transmembrane proteins, with a heterodimeric structure consisting of the Notch extracellular domain (NECD), a single transmembrane domain, and the Notch intracellular domain (NICD) (Figure 1) [26]. The NECD of the receptors in mammals contains approximately 26-36 epidermal growth factor (EGF)-like domains, and three cysteine rich repeats, known as Lin repeats. These domains enable ligand-receptor interaction. For instance, the interaction of delta-like ligands of a neighbouring cell with EGF 11 and 12 of the extracellular domain activates the Notch signalling pathway [27] Ligand binding induces cleavage mediated by the enzyme complex ℽ-secretase [18]. Following proteolytic cleavage, further activation of Notch signalling is mediated by the NICD, whose nuclear translocation changes recombining binding protein suppressor of hairless (RBPJ) proteins to transcription activators. Together with Master mind-like polypeptides, this Notch transcription activation complex regulates downstream gene expression [28]. While the activation mechanisms of the Notch pathway appear well described, the distinct functions of each receptor remain poorly understood. Studies attempting to compare the structure and 3
function of the Notch receptors progressively highlight distinctive features of the Notch-3 receptor that may explain its emerging key role in tumorigenesis. For instance, although the HES-1 transcription factor is the most commonly known Notch target (Figure 1) [29, 30], the Notch-3 receptor appears a poor activator of specific HES-1 target genes. Transfection of the Notch-3 NICD in the human chorion carcinoma JEG cell line failed to trigger HES-1 promoter activity irrespective of the concentration of RBP-Jk available, whereas Notch-1 NICD overexpression yielded strong activation of the promoter [31, 32]. Further evidence suggest that Notch-3 and Notch-4 diverge from the known structure of the Notch-1 and Notch-2 receptors [33]. All Notch receptors carry a PEST sequence near the C-terminus domain involved in negative regulation of the pathway and the degradation of the NICD in the nucleus [32]. This sequence is located beside the Notch transcriptional activation domain (TAD), which is capable of autonomous transcriptional activation [34]. The TAD shows substantial evolutionary divergence among the four mammalian Notch receptors. But unlike other Notch receptors (Notch-1, Notch-2), Notch-3 contains both TAD and cytokine response regions (NCR) post cleavage [35, 36]. This particularity could explain the ability of Notch-3 to repress HES-1 signalling [37, 38] A frameshift mutation in the Notch-3 receptor was identified at amino acid position 1802 [39] and upstream the PEST domain of the receptor in triple negative breast cancer [40]. Mutations in Notch receptor genes are common and were present in the majority of the 905 tumour lines examined by Mutvei et al., with 5% of cell lines carrying more than one mutation [39]. Frameshift mutations were the second most commonly observed mutations in Notch receptors [39]. In this extensive dataset, the type and frequency of these mutations varied according to tumour types. Cell lines derived from tumours of the prostate and endometrial cells presented the highest frequency in Notch mutations. The oesophagus and urinary tract contained considerable less mutations but still at a significant frequency of greater than 25% [39]. The mutation frequency of Notch was determined to fall below that of Ras, but within a similar range to the ErbB1-4 and APC genes [39]. The range of frequencies when examining the different receptors and ligands of the Notch pathway varied from 7.3% in Notch-1, down to 2.9% in Delta-like ligand 4. [39] The significance of this unique Notch-3 mutation is yet to be elucidated [39] but is known to result in the loss of the functioning PEST domain at the Cterminus end of the protein [39, 40]. PEST is involved in the tight regulation of the ICD domain and deletion increases stability and half-life of the NICD [41]. This may explain over expression of the signalling pathway in cancer cells [41].
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Notch-3 receptor: a driver of the malignant phenotype The clinical evidence for the role of Notch-3 overexpression when compared to benign tissue as a driver of malignancy is rapidly accumulating across a variety of cancer sites. In the cervix, immunochemistry analysis of 168 biopsy specimens identified increasing NOTCH-3 staining intensity with increasing disease stage: 86.7% (26/30) of pre-cancer and 85.7% (84/98) of analysed tumour tissues showed significant cytoplasmic and nuclear NOTCH-3 positivity. In pre-cancerous lesions, the presence of high cytoplasmic NOTCH-3 staining was dominant in the 87.5% of specimens testing positive for human papilloma virus (HPV)-16 [42]. In breast cancer, in situ mRNA hybridisation identified high NOTCH-3 expression in 6 of the 50 tumours profiled by Reedijk et al. [43], which correlated with increased predicted mortality at 10 years. In this small sample, these tumours were almost exclusively triple negative (negative for ErbB2, and both the estrogen and progesterone receptors expression). In inflammatory breast cancer patients, elevated nuclear expression of NOTCH-3 was detected in 80% of the 25 cases tested [44]. More recently mechanistic studies using antagonist and agonist Notch-3 antibodies demonstrated that Notch-3 activation promotes the development of basal breast cancer [45].
In lung cancer, NOTCH-3 immunostaining intensity was highest in non-small cell (NSCLC) patient biopsy specimens (adenocarcinoma (n=24); squamous cell carcinoma (n=20)) and lowest small cell carcinoma (n=13) when compared to neighbouring normal tissue [46]. This result was confirmed by a meta-analysis of 19 studies involving 3663 NSCLC patients [47]. This work further identified that NOTCH-3 expression in NSCLC tissues significantly correlates with the overall survival rate of NSCLC patients. The overexpression of both Notch-3 mRNA and immunostaining levels was reported in tongue squamous cell carcinoma patient specimens. Expression was elevated in the majority of the 74 specimens profiled, but failed to correlate with clinical staging [48, 49]. Fibroblasts in the cancer stroma, especially those adjacent to the tumour periphery, showed positive staining for NOTCH-3 in 31 out of 93 cases examined by immunochemistry [50]. In ovarian cancer, high mRNA expression level of Notch-3 was associated with better overall survival for all cancers but the serous and endometrioid subtypes [51, 52]. NOTCH-3 mRNA levels and immunostaining intensity were elevated in intestinal-type carcinomas [53] Elevated NOTCH-3 NICD expression levels were elevated in tumour-bearing bones compared to control bones in a murine model of multiple myeloma [54]. In gallbladder malignancy, increased NOTCH-3 immunostaining intensity was associated with larger tumour size, invasion, metastasis and low curability rates in both
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squamous cell/adenosquamous carcinomas (n=46) and adenocarcinomas (n=80) [17]. Zhang et al. identified elevated NOTCH-3 mRNA levels in over 71.8% of the 32 cases of hepatocellular carcinomas studied which positively correlated with alpha-fetoprotein but negatively correlated with both differentiation grade and survival [55]. In glioblastoma, NOTCH-3 was moderately upregulated in 40 % of the 15 samples tested [56]. In these disease, NOTCH-3 amplification was associated with significantly worse outcome (median survival 10 vs. 28 months, Log-Rank test ) compared to tumours with non-amplified locus in the 60 tumour biopsies analysed. The amplification resulted in a significant elevation of NOTCH-3 protein levels, when compared to samples with non-amplified locus [57] . An increase in NOTCH-3 expression was also reported in melanoma specimens, possibly enabling differentiation between malignant melanoma and benign Nevi [58]. In a study carried out by Man et al., NOTCH-3 was expressed in 92.5% of patients with Epstein-Barr Virus (EBV) positive tumours and all other nasopharyngeal tumours, demonstrating that Notch-3 was heavily implicated in the development of nasopharyngeal cancer [59]. But conflicting data have been reported in regards to the implication of Notch-3 in renal malignancies [60]. In a cohort of 80 patients analysed by tissue microarray, the presence of NOTCH-3 was better correlated with renal carcinoma when the latter was present in the right kidney [60]. Further analysis of 84 cases detected low level positivity for NOTCH-3 staining in renal tumours staged T1 or T2 but the results were not statistically significant [61]. Finally, the elevation of Notch-3 mRNA and immunostaining intensity was documented in prostate cancer. Although one study reported that NOTCH-3 was not expressed in the metastatic prostate cancer cell lines LNCaP, PC-3 and PC-3M) [62], another identified high expression of both HES-6 and NOTCH-3 in VCaP and LnCaP96 cells and proposed that these two markers could identify aggressive prostate cancers [63]. Further reports supporting the involvement of Notch-3 in prostate tumorigenesis exist. Notch-3 has a critical role to play in the development of the prostate gland as well as prostate cancer [64]. NOTCH-3 expression was positively correlated with the grading of prostate cancer [65, 66]. Furthermore, high levels of NOTCH-3 post-radical prostatectomy were indicative of recurrence [67].
A reduction in Notch-3 expression can however also be indicative of malignancy. In lung cancer specimens, NOTCH-3 expression in the cancer tissue was weaker than that of the corresponding non-tumor tissue in the SCLC group [46]. This also appears the case in thyroid carcinoma. Medullary thyroid carcinomas patient specimens analysed were deficient in NOTCH-3 intracellular domain 6
while high levels were detected in normal thyroid epithelial cells [68]. Analysis of a tissue microarray of normal and pathologic thyroid biopsies from 155 patients identified that NOTCH-3 expression levels regressed across decreasingly differentiated, increasingly malignant thyroid specimens and were predictive of survival [69].
Notch-3 receptor: a molecular switch The detailed evaluation of the molecular consequences of Notch-3 activation using siRNA, neutralising antibodies or Notch inhibitors is under scrutiny. As expected, Notch-3 appears directly implicated in proliferation and invasion of cancer cells (Figure 1). In MCF-7 breast cancer cells, increased expression of the Notch-3 N3ICD was associated with arrest of cell cycle at the G0/G1 phase [70], but in Erb B2-Negative tumour cell lines down regulation of Notch-3 lead to decreased proliferation and increased apoptosis [71]. As a result Notch-3 targeted therapies may be of most benefit for the treatment of Erb B2-negative breast tumours [71, 72]. Activation of Notch-3 was additionally associated with the upregulation of IL-6 in breast cancer cells [73]. IL-6 has already been established as a major mediator of inflammatory responses in humans [74], and a critical player within the pathophysiology of cancer. [73].
In NSCLC, manipulation of Notch-3 expression lead to a modification in the expression levels of NOTCH-1, HES-1 and JAGGED-1 [46], and suggestions of its regulation by Protein Kinase Ci [75]. Characterisation of the impact of Notch-3 manipulation on cell adhesion, motility and EpithelialMesenchymal Transition in vitro concluded that Notch-3 could act as a tumour suppressor in NSCLC but as a tumour promoter in SCLC [46]. Notch-3 inhibition using siRNAs prevented cell proliferation in NSCLC cell lines. In SCLC, Notch-3 inhibition stimulated cell proliferation [46, 76]. Similarly, treatment of follicular thyroid cancer cell lines with the histone deacetylase inhibitor AB3 increased NOTCH-3 NICD levels, decreased the expression of neuroendocrine tumour markers (ASCL 1 and CgA) and affected cell proliferation [68]. In vivo, NOTCH-3 NICD was essential to the reduction of tumour burden through apoptosis [69]. In prostate cancer cells, the regulation of NOTCH-3 by p38 MAPK, including its mRNA half-life time, was key to the induction of luminal differentiation [77]. In T-ALL, the tumorigenic properties of Notch-3 may be mediated through the regulation of miR-223 and the downstream inhibition of the tumour suppressor FBXW7. This mechanism may be key to the response of T-ALL to gamma-secretase inhibitor (GSI) treatment, as miR-223 inhibition prevented TALL resistance to these agents [78]
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The biological impact of Notch-3 deregulation is associated with the induction of chemoresistance (Figure 1). Patients with advanced NSCLC whose tumours overexpressed NOTCH-3 were at a higher risk of resistance and poor overall survival following platinum-based chemotherapy [79]. These tumour specimen also overexpressed the cancer stem cell markers ALDH1A1 and CD44. Blockade of Notch-3 using siRNAs in A459 lung cancer cells lead to a reduction in LC3II expression and prevented autophagy activation [79, 80]. In nasopharyngeal cell lines, increased sensitivity to cisplatin treatment following the suppression of Notch-3 by siRNAs was also demonstrated [59]. In breast cancer cells, high NOTCH-3 expression correlated with reduced chemoresistance [81], due to the negative regulation of the drug efflux pump P-glycoprotein by NOTCH-3. The study additionally proposed the epigenetic silencing of NOTCH-3 through methylation as an underlying mechanisms of chemoresistance in breast cancer [81]. In triple negative breast cancer patients, high coverage whole genome sequencing together with transcriptome and whole exome sequencing identified that structural variations are associated with the amplification of NOTCH-3. But the receptor appears particularly important in the chemoresponse of ovarian cancer cells [82, 83]. Chemoresistant ovarian cancers were associated with a higher level of NOTCH-3 than NOTCH-1 [83]. In vitro, silencing of NOTCH-3 lead to improved sensitivity of ovarian cancer cells to paclitaxel [83]. The induction of miR136 in ovarian cancer cells repressed Notch-3 mediated signalling and re-sensitized paclitaxelresistant ovarian cancer cells, likely through a reduction in cancer stem cells (CSC) activities [84]. The Notch pathway is well documented to promote the growth of CSCs and thereby contribute to the resistance of tumours to chemotherapy and radiotherapy [85]. In breast cancer, often referred to as a “stem cell disease”, Notch-3 and its associated active pathway has been attributed a role in the self-renewal of CSCs, and the promotion of cell survival under hypoxic conditions [73]. In hepatoma cell lines, down regulation of NOTCH-3 affected sensitivity to cisplatin in a mechanisms proposed to involve the interaction of Notch-3 with the Wnt/beta-catenin pathway to regulate stemness [55]. Similarly, the downregulation of NOTCH-3 using lentilvirus in melanoma cell lines resulted in a reduction in the expression levels of stem cell markers, such as CD113 and CD271, and severely compromised the tumorigenicity of the cells in vivo. Taken together this clear association between Notch-3 expression and the CSC population strengthen the need to evaluate Notch-3 targeted therapeutic approaches. These may be of particular relevance to prevent undesirable effects of other molecular approaches. Indeed, treatment of EGFR-mutated lung cancer cell lines with the EGFR tyrosine kinase inhibitor erlotinib was associated with an enrichment of the ALDH(+) stem-like cell population through EGFR-dependent activation of Notch-3, likely compromising treatment efficacy [86]. Similarly, the inhibition of NOTCH-2 by gliotoxin in chronic lymphocytic leukaemia (CLL) cells was associated with an increase in NOTCH-3 expression [87]. 8
Notch-3 receptor: a driver of vascular expansion Components of the Notch system were reported to be expressed at different levels during vascular development and therefore, its role in vascular expansion during tumorigenesis is likely. The angiogenetic properties of Notch-3 has been recently investigated by Liu H et al. The authors described Notch-3 as a critical regulator of blood vessels formation in both a developmental phase and pathological event [88]. In triple negative breast cancer, microvascular density correlated with elevated Notch-3 mRNA and protein levels, but not those of the vascular endothelial growth factor, suggesting Notch-3 as a potentially key therapeutic target of neovascularization [89]. In liver cancer, immunohistochemical analyses of patient specimens revealed that only the increased level of NOTCH-3 was positively correlated with vascular invasion [90]
Notch-3 stimulates the growth of vascular smooth muscle cells via inhibition of a cycline dependent kinase [91]. The receptor is highly expressed in the response to vascular damage [91], possibly through the action of miR- 206 [92]. The exact function of Notch-3 in relation to these vascular events remains poorly characterised [93], but a role for Notch-3 in lung hypertension is emerging [94]. Aberrant Notch signalling was noted within multiple other pathological processes, including the degenerative disease CADASIL [16], Alagille syndrome and spondylocostaldysostosis [95].
The role of Notch-3 in the vasculature highlights the potential key relevance of this receptor to the cellular adaptation of cancer cells to hypoxia. We have proposed this link within the context of prostate cancer [96] and demonstrated, like others [97, 98], that Notch-3 expression may be elevated in response to hypoxic exposure and that hypoxic conditions may alter the activation of Notch-3 mediated signaling [99]. In breast cancer cells, Notch-3 activity was essential to the induction of carbonic anhydrase 9 (CA9) under hypoxic conditions [100]. The interaction of CA9 with miR-34a, which acts as a feedback loop to the modification of NOTCH-3 levels, was proposed to participate in the regulation of human mammospheres under these conditions [101].
But the role of Notch-3 could potentially further lie in the regulation of tumour dormancy (Figure 1) [102]. In T-ALL and colorectal cancer cells, escape from dormancy was associated with an increased in the expression of the Notch ligand DLL4 and the Notch-3 receptor [103]. The interaction between DLL4 and Notch-3 may repredent a key molecular switch to the promotion of tumorigenicity [104], which leads to the regulation of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1)
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levels [102]. In breast cancer, this mechanisms could drive recurrence following HER2/neu pathway inhibition [105].
Notch-3 targeted therapies The ability to control the intensity of Notch signalling has high therapeutic relevance [106] and a number of therapeutic approaches are currently under development. These include gammasecretase inhibitor, Alpha-secretase inhibitor and immunotherapy with neutralising antibodies (Figure 1) [106-110] C-secretase is a protein complex that is activated by the binding of Notch ligands (Jag 1, Jag 2, and DLL 1-3) to Notch receptors. It was identified in 1999 and the therapeutic potential of its inhibition has been under intense scrutiny in many cancer sites [111]. An increase sensitivity of cancer cells to cytotoxic chemotherapy and radiotherapy can be achieved with the use of gamma secretase inhibitors (GSI). This effect may be in part dependent on the induction of apoptosis by Notch-3 in endothelial cells [112]. In ovarian cancer cell lines, sensitisation to paclitaxel was of the same magnitude following GSI treatment or Notch-3 knockdown [83]. This suggests Notch-3 targeting as a strong approach to inhibit Notch signalling and possibly reduce the known side effects of GSIs. In NSCLC, combination of GSIs with radiation prevented radiation-induced increase in NOTCH-3 expression and increased cellular radio sensitivity [113]. In glioblastoma tumour-initiating cells, 17 genes representative of active Notch signalling components including NOTCH-3, identified in 49.3% of the tumours profiled in The Cancer Genome Atlas expression dataset, discriminated good from poor responder to GSI treatment [114]. This data suggests that pre-treatment screening of Notch activity could identify the subgroup of patients most likely to respond to treatment.
The second class of agents considered for Notch inhibition are monoclonal antibodies [115]. A more recent example of this group of inhibitors is the antibody OMP-59R5,[116] which targets both Notch3 and Notch-2 receptors. The antibody, alone or in combination with chemotherapy, proved efficient in inhibiting the expression of Notch target genes and reducing tumour-initiating cell frequency in a broad spectrum of epithelial tumours, including breast, lung, ovarian, and pancreatic cancers [116]. The antibody which just recently entered phase 1 trials for dosage treatment in patients, was developed to specifically target the Notch-2 receptor and consequently prevent ligand binding. In further studies it was observed that the antibody also binds with Notch-3 and further prevents interactions with this specific receptor and its associated ligands. Those treated with the drug were showed non-linear clearance of tumours present from pancreatic cancer, with diarrhoea reported as the most extreme side effects of the treatment [116]. A similar approach is showing exciting results 10
in NSCLC. In cell line models and patient-derived xenograft tumours, the dual targeting of EGFR and Notch2/3 receptors with the antibody CT16 prevented acquired resistance to EGFR inhibitors and radiation through the reduction of the cancer stem cells compartment [117]. These approaches may be particularly important for the management of tumours with Notch-3 mutations. Bernasconi-Elias P et al [41] identified that mutation of the NRR domain of the Notch-3 receptor in T-ALL cell lines resulted in an increase in ligand-dependent Notch-3 signalling. This mutation is associated with a chemical substitution where a hydrogen bond initially formed between a backbone nitrogen and oxygen side chain is broken. This weakens the interaction between the nitrogen and oxygen side-chain, both which are located close to the S2 site of cleavage in the receptor. This weakened interaction possibly makes the S2 site more accessible by proteases to cleave the NICD domain from the receptor. This hypothesis is supported by the elevated levels of NOTCH-3 NICD in cell T-ALL cell lines [41]. The growth and survival of cancerous cell lines such as TALL proved to be dependent on this specific mutation. Upon the addition of an inhibitory antibody of the Notch-3 receptor in mice, tumour growth was compromised [41]. Pre-treatment determination of Notch-3 mutational status may thus represent a key step in the identification of the subset of patients most likely to benefit from Notch-3 targeted therapy. But other strategies are emerging. Emerin was proposed as a potential target within the Notch pathway [118]. This protein has the ability to down regulate Notch signalling. The inner membrane protein induces retention of the NICD once cleaved from the Notch receptor, preventing entrance into the nucleus to activate target genes. Emerin thus represent a prime therapeutic target to selectively control the activation of Notch signalling [118]. Similarly, the possible interaction of the pathway with survivin, an inhibitor of apoptosis abnormally expressed cancer, represent an attractive strategy to Notch targeting [119].
Conclusion The evidence for the role of the Notch-3 receptor in tumorigenesis is rapidly accumulating. This Notch receptor and the pathway in whole have been specifically targeted in the hopes of developing a successful anti-cancer treatment. There has already been positive results demonstrating significant tumour regression when effectively “switching off” the Notch pathway. Further evaluation of the exact impact of Notch-3 mediated signalling on tumorigenesis is warranted and will likely guide the development of more potent, specific, anti-cancer agents.
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Figure1: Schematic representation of the activation and downstream functions of the Notch-3 receptor in tumorigenesis. (1) The interaction of Notch ligand with the notch extra cellular domain (NECD) of the receptor leads to the cleavage of the receptor by the gamma-secretase enzyme, and the release of the notch intra-cellular domain (NICD). The nuclear translocation of the NICD and its interaction with the transcription factor RBPJ (recombining binding protein suppressor of hairless) enables the induction of the expression of downstream Notch-3 target genes, but not HES-1. (2) Activation of Notch-3 leads to the induction of a variety of biological processes driving tumorigenesis, including angiogenesis and the release from tumour dormancy. (3) The biological functions of Notch-3 signalling participate in chemoresistance in many cancer types, such as ovarian and lung cancer; promote metastasis and a poor patient prognosis (e.g non-small cell lung cancer (NSCLC), ovarian and glioblastoma). (4) The increasingly described role of Notch-3 in tumorigenesis warrants the investigation of Notch-3 inhibition therapeutic strategies. Gamma secretase inhibitors and antibodies targeted against the receptor are already yielding promising results. (+) denote a positive action of Notch-3, (-) a repressive action.
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Notch ligands Jagged 1,2, DLL 1-4
Elevated expression in most cancers.
4 NECD
Notch-3
Frameshift mutations.
Gamma secretase
Inhibitors
NICD
Extra-cellular space
Cytoplasm
Cleavage
1
TAD and NCR present
NICD
NICD
RPBJ
-
Nucleus
+
Notch-3 target genes
HES-1
Nucleus P300 / RBPJ
2
Cell migration
Proliferation
Differentiation
Apoptosis
Lung + cancer
Autophagy
P-glycoprotein
Breast + cancer
IL-6
Angiogenesis
+ -
-
SCLC NSCLC
Tumour dormancy
3 Chemoresistance + Ovarian, lung cancer
Metastasis
Poor prognosis
Conflict of interest
All authors have no conflict of interest to declare.
Highlights
Deregulation of Notch-3 is associated with more aggressive, often chemo resistant malignancies
Notch-3 acts as a molecular switch to a variety of biological processes including angiogenesis and the release from tumour dormancy.
Therapeutic strategies against Notch-3 are showing promise in preventing tumour growth alone or in combination with chemo- or radio- therapy.
19