Review

Oncogene (2008) 27, 5124–5131; doi:10.1038/onc.2008.226

Rational targeting of Notch signaling in cancer

P Rizzo1, C Osipo1, K Foreman1, T Golde2, B Osborne3 and L Miele1

  1. 1Breast Cancer Program, Cardinal Bernardin Cancer Center, Loyola University Chicago, Chicago, IL, USA
  2. 2Department of Neurology, Mayo Clinic at Jacksonville, Jacksonville, FL, USA
  3. 3Department of Veterinary and Animal Sciences, University of Massachusetts at Amherst, Amherst, MA, USA

Correspondence: Professor L Miele, Breast Cancer Program, Cardinal Bernardin Cancer Center, Loyola University, 2160 South First Avenue, Room 236, Maywood, Chicago, IL 60163, USA. E-mail: lmiele@lumc.edu

Top

Abstract

Accumulating preclinical and clinical evidence supports a pro-oncogenic function for Notch signaling in several solid tumors, particularly but not exclusively in breast cancer. Notch inhibitory agents, such as γ-secretase inhibitors, are being investigated as candidate cancer therapeutic agents. Interest in therapeutic modulation of the Notch pathway has been increased by recent reports, indicating that its role is important in controlling the fate of putative ‘breast cancer stem cells’. However, as is the case for most targeted therapies, successful targeting of Notch signaling in cancer will require a considerable refinement of our understanding of the regulation of this pathway and its effects in both normal and cancer cells. Notch signaling has bidirectional ‘cross talk’ interaction with multiple other pathways that include candidate therapeutic targets. Understanding these interactions will greatly increase our ability to design rational combination regimens. To determine which patients are most likely to benefit from treatment with Notch inhibitors, it will be necessary to develop molecular tests to accurately measure pathway activity in specific tumors. Finally, mechanism-based toxicities will have to be addressed by a careful choice of therapeutic agents, combinations and regimens. This article summarizes the current state of the field, and briefly describes opportunities and challenges for Notch-targeted therapies in oncology.

Keywords:

Notch, cancer, targeted therapy

Top

Introduction: why target the Notch pathway?

For those of us involved in the pharmacological treatment of human cancers, whether experimental or standard of care, the most frustrating feature of neoplastic diseases is the inherent ability of cancer cells to respond to therapy by adapting and/or selecting resistant subclones. In clinical practice, this results in drug-resistant recurrence of disease, often in a metastatic form even after apparent eradication. Much of the morbidity and mortality from cancer is the result of such recurrences.

For decades, we have been searching for a ‘magic bullet’ that could hit an elusive target only cancer cells depend upon. Yet, to date most of pharmacological cancer treatments still rely on the relatively blunt instruments of traditional chemotherapy. Targeted agents are slowly but steadily being added to our therapeutic arsenal. These are directed largely at growth factor receptors or their associated kinase activities, as well as angiogenic growth factors. Monoclonal antibodies (mAbs) such as trastuzumab, pertuzumab, rituximab, bevacizumab and others, as well as tyrosine kinase inhibitors, such as imatinib, gefitinib, lapatinib and others have gained clinical acceptance as part of combination regimens. Some of these agents have proven to be remarkably effective. Yet, even with these agents resistance is observed.

Our increasing understanding of cancer biology is beginning to explain the reasons for these therapeutic failures (Hanahan and Weinberg, 2000). First, decades of signal transduction research have revealed that the receptors, enzymes and transcription factors that regulate cell fate are virtually all connected into an amazingly complex network of cross-regulatory interactions. The Internet rather than an old-fashioned machine may be an apt metaphor for the workings of cell signaling. Even if one or several ‘routers’ are taken out of commission, Internet traffic can be rerouted around them. To completely stop Internet traffic, a very large number of routers or cables need to be disabled. Second, we have learned that the cell-fate control system is not only interconnected but also highly redundant, such that if a gene or protein is disabled, another can perform a similar function. Connectivity and redundancy mean that if the system is stressed (for example, by a targeted drug that blocks a growth factor receptor), the system can reset itself to a new status that works around the block or replaces the disabled receptor with a similar one. All this makes evolutionary sense. Eukaryotic cells are very complex and delicate objects with tens of thousands of moving parts. To allow survival in an often forbidding environment, evolution has created a robust system of cell-fate regulation that is highly resistant to the loss of one or even a few components. Unfortunately, this adaptability is hijacked by cancer cells, making the search for ‘magic bullets’ a very frustrating task. Even though cancers can become ‘addicted’ to specific pathways, they are remarkably adept at weaning themselves from their addictions if the need arises, and find alternatives to pathways hit by therapeutics. This is, in part, because the genomic instability resulting from loss of DNA repair checkpoints, in transformed cells makes these cells capable of selecting drug-resistant mutants by utilizing the built-in connectivity and redundancy in signaling. Thus, the reason why ‘magic bullets’ have been so hard to find is that there are very few, if any, individual, irreplaceable targets that are uniquely associated with cancer cells.

Is there any hope of finding an Achilles' heel in cancer? Perhaps. Not all pathways are created equal. Some of the most ancient cell-fate control pathways which evolved with the initial appearance of multicellular organisms are conserved in all living organisms. These pathways function as ‘Internet service providers’ in the signaling network. That is, they are multifunctional and control key nodes in the traffic of signaling information that regulates differentiation, proliferation and survival. The Wnt, Hedgehog and Notch pathways belong to this ‘aristocracy’ of signaling systems. Not surprisingly, they are important in developmental biology and come into play whenever critical cell-fate decisions are made. Learning how to safely and effectively manipulate these pathways in cancer cells may bring us the next quantum leap in cancer therapy. The Notch pathway, the subject of this article, is a short-range communication system in which contact between a cell expressing a membrane-associated ligand and a cell expressing a transmembrane receptor sends the receptor-expressing cell (and quite possibly both cells) a cell-fate regulatory signal. This signal takes the form of a cascade of transcriptional regulatory events that affects the expression of hundreds if not thousands of genes, and has profound phenotypic consequences that are context dependent. The basic features of the pathway and the possible biological roles of Notch signaling in human malignancies have been discussed in several recent reviews (Allenspach et al., 2002; Miele, 2006; Miele et al., 2006; Berman and Look, 2007; Koch and Radtke, 2007; Roy et al., 2007; Shih and Wang, 2007). In most cases, its deregulation has oncogenic effects, with the notable exception of epidermal keratinocytes where Notch-1 functions as a tumor suppressor. In several malignancies going from T-cell acute lymphoblastic leukemia (Roy et al., 2007) to breast cancer (Reedijk et al., 2005; Dickson et al., 2007) to melanoma (Pinnix and Herlyn, 2007) to lung adenocarcinoma (Chen et al., 2007) and others, the inappropriate activation of Notch signaling results in signals that stimulate proliferation, restrict differentiation and prevent apoptosis in cancer cells. As a result, Notch signaling inhibitors are being actively investigated for the treatment of a variety of malignancies.

An additional reason for focusing on Notch and other ancient developmental pathways is that in recent years, a distinct cellular hierarchy has been identified in hematopoietic and some solid tumors. Many cancers appear to contain a small population of pluripotent ‘stem cells’ or ‘tumor-initiating cells’ that give rise to the bulk population of cancer cells through a process of aberrant differentiation that recapitulates that of normal tissues (Song and Miele, 2007). These ‘cancer stem cells’ are characterized by properties of normal stem cells, such as indefinite self-replication through asymmetric cell division, very slow proliferation rates and resistance to toxic agents due in part to high-level expression of ABC transporters. Whether cancer stem cells are derived from the malignant transformation of normal tissue stem cells or from the ‘dedifferentiation’ of normal non-stem cells is a matter of considerable debate. What seems likely is that these cells are uniquely capable of resisting anticancer agents, surviving for a long time in a nearly quiescent status and produce recurrences and metastases. Thus, a complete eradication of these cells will be necessary to attain a cure. This will require targeting of pathways that participate in the survival, replication and differentiation decisions in undifferentiated, pluripotent cells, such as the Wnt, Hedgehog and Notch pathways (Song and Miele, 2007). Indeed, there is significant evidence that Notch is relevant to the survival of breast cancer ‘stem cells’ (Farnie et al., 2005, 2007; Farnie and Clarke, 2007; Sansone et al., 2007).

Finally, the interaction between cancer cells and the surrounding stroma is receiving increasing attention as a key factor in tumor progression. ‘Tumor stroma’ includes endothelial cells, necessary for tumor angiogenesis, fibroblasts that can produce growth factors and cytokines, as well as many subtypes of immunocytes, from T cells to dendritic cells to NK cells that can affect tumor progression either favorably or unfavorably. There is significant evidence that bidirectional intercellular communication involving Notch signals takes place between tumor cells and stromal cells in some malignancies (Jundt et al., 2002, 2004; Houde et al., 2004; Zeng et al., 2005), suggesting that targeting the Notch–ligand interaction in endothelial cells can have therapeutic applications (Yan and Plowman, 2007). Moreover, Notch signaling has multifaceted functions in the immune system that need to be taken into account when planning therapeutic interventions (Dallman et al., 2005; McKenzie et al., 2005; Minato and Yasutomo, 2005; Minter et al., 2005; Tu et al., 2005). This article discusses current efforts to develop Notch-targeted cancer therapeutics by both small molecules and biologics, including pros and cons of different targeting strategies as well as identifying challenges to overcome.

Top

Targeting Notch in cancer

For targeting purposes, some features of the Notch pathway have unique relevance. First, the fact that the signaling cascade triggered by Notch–ligand interactions does not include an enzymatic amplification step (for example, a nucleotide cyclase or a kinase) means that ‘signal intensity’ can be modulated very precisely by cellular regulatory mechanisms. As a result, the downstream effects of Notch activation are exquisitely dose dependent (Miele et al., 2006). This means that complete shutdown of the pathway may not always be necessary to achieve a therapeutic effect. A second key feature is that the intracellular half-life of the active form of Notch is generally very short, in the order of minutes, though it may be longer in transformed cells (Weijzen et al., 2002). The Notch signal is essentially a short pulse of gene regulation (Miele et al., 2006). This implies that sustained inhibition may not be always necessary and that intermittent inhibition may be successful. A third important feature to keep in mind is that the effects of Notch are remarkably context dependent. This means that Notch signals can be used for different purposes in different cell types, and for each cell type the effects of Notch manipulations need to be investigated without preconceived assumptions. Systemic inhibition of Notch signaling is likely to have a multitude of effects in different cell types. Thus, for therapeutic purposes we shall have to determine whether there is a level (or timing) of Notch inhibition that is sufficient to attain efficacy in disease control without causing intolerable adverse effects. An alternate strategy may be to identify more context-specific targets within the Notch pathway, selectively effective drug combinations (based on cell-specific cross talk) or designing more selective delivery strategies for Notch inhibitors.

Inhibition of Notch signaling can be achieved theoretically at many different levels. Agents targeted to some of these levels have been described, whereas others remain hypothetical possibilities (Figure 1). It is possible to interfere with Notch–ligand interactions by using receptor decoys (Nickoloff et al., 2002), blocking ligand ubiquitination/trans-endocytosis (Pitsouli and Delidakis, 2005) or Notch receptor fucosylation (Okajima and Irvine, 2002). It is possible to interfere with receptor activation by blocking ligand-induced conformational changes in Notch receptors (Gordon et al., 2007), receptor cleavage by ADAM proteases (Brou et al., 2000) or γ-secretase (Kopan and Ilagan, 2004; Miele et al., 2006), as well as Notch monoubiquitination (Gupta-Rossi et al., 2004). Finally, it is conceivable that Notch signaling could be inhibited by disrupting protein–protein interactions involved in Notch-dependent nuclear events (Nam et al., 2006, 2007), including assembly of co-activators with the Notch transcriptional complex (NTC) and formation of higher order DNA-bound complexes. As of this writing, γ-secretase inhibitors (GSIs) are in early clinical trials in various institutions, and mAbs that ‘lock’ Notch receptors in an inactive conformation by binding to the ‘negative regulatory region’ (NRR) are in preclinical development (Li et al., 2008). Moreover, mAbs that target Notch ligand DLL4 (Ridgway et al., 2006) have been shown to inhibit Notch signaling in endothelial cells and cause disorganized angiogenesis. These mAbs are currently being developed as antineoplastic agents (Noguera-Troise et al., 2007; Thurston et al., 2007; Yan and Plowman, 2007).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Diagram of putative therapeutic targets in the Notch pathway. (Red) Flat-tipped arrows indicate inhibitors. (Green) Sharp-pointed arrows indicate stimulation of Notch degradation by the Numb pathway (e.g., by inducing Numb expression) or the β-arrestin/Kurz pathway (e.g., by inducing expression of β-arrestin or Kurz). Asterisks indicate targets for which specific agents have been developed to date. Question marks indicate potential targets, yet unverified. Theoretically, inhibition of Notch signaling could be achieved by targeting ligand ubiqutination (UQ), ligand-receptor interaction, NEC/NIC dissociation, a disintegrin and metalloprotease (ADAM)-mediated cleavage, receptor ubiquitination/endocytosis, γ-secretase cleavage, assembly of the co-activator complex with Notch and C-promoter binding factor 1 (CBF-1) and heterodimerization of DNA-bound Notch transcriptional complexes (NTCs). In addition, expression of receptors and ligands as well as expression of selected downstream Notch targets could be conceivably targeted (e.g., by modulating pathways that regulate Notch or ligand expression, or by using RNA-targeted therapies to modulate translation of Notch targets.

Full figure and legend (175K)

Each of these approaches has potential advantages and disadvantages. GSIs are active in several experimental models and have the advantage of relative ease of administration, oral bioavailability and low cost. In general, small molecules can be dosed more precisely than biologics because of their relatively short biological half-life and simpler dose–response relationships, and this may be important in a field where the therapeutic window may be small. An additional potential advantage is the fact that a single agent can block the activation of all four Notch homologues. In our experience and that of others, some solid tumors such as breast cancers and melanomas co-express several Notch homologues, and it is conceivable that redundancy between them could blunt the effects of more selective inhibitors and lead to resistance. On the other hand, there is some evidence that different Notch homologues may have opposite effects. For instance, Notch-2 appears to counteract the pro-oncogenic effects of Notch-1 and Notch-4 in breast cancer cells (O'Neill et al., 2007), and its expression correlates with better differentiated tumors (Parr et al., 2004) unlike that of Notch-1 and Jagged-1, which correlate with poor prognosis (Reedijk et al., 2005; Dickson et al., 2007). Similar differences have been noted in embryonal brain tumors (Fan et al., 2004), where conversely it is Notch-2 that has an oncogenic role. Another obvious potential disadvantage of GSIs is off-target effects. Most GSIs in development are competitive inhibitors that mimic the structure of short hydrophobic peptides. Given the fact that γ-secretase has numerous targets other than Notch receptors and a rather promiscuous cleavage specificity (Kopan and Ilagan, 2004), it is to be expected that GSIs will not specifically inhibit the cleavage of Notch receptors. It should be noted that off-target effects are the rule rather than the exception for most approved small molecule drugs. The mere fact that an agent can have off-target effects should not discourage its therapeutic development, unless it can be demonstrated that a more selective agent offers safety or efficacy advantages. The recent discovery that some nonsteroidal antiinflammatory drugs and structurally related compounds can allosterically modify the substrate specificity of γ-secretase and decrease or increase the production of Aβ42 amyloid peptide (Kukar and Golde, 2008) raises the prospect that a class of γ-secretase modifiers (GSM) can be developed that are capable of selectively modulate the cleavage of Notch receptors as opposed to other substrates, though specificity for individual Notch homologues is unlikely.

Is there a role for biologics (mAbs or recombinant decoys) in Notch modulation? If specificity for an individual receptor or ligand is desirable, biologics are more likely to deliver such specificity than small molecules. Particularly mAbs, if epitopes are carefully selected, can achieve exquisite specificity and very high affinities. One situation in which a specific biologic may be preferable is if the target has a relatively restricted expression pattern compared to other Notch pathway members and/or can be replaced through redundancy in nontarget tissues. This may limit mechanism-based toxicities. For example, the expression pattern of Notch-4 appears to be considerably more restricted than that of Notch-1, suggesting that agents targeting Notch-4 may have less systemic toxicity than agents targeting Notch-1. The mAbs to DLL4 (Noguera-Troise et al., 2007; Thurston et al., 2007; Yan and Plowman, 2007) represent a promising approach in this direction. These mAbs exploit the role of DLL4–Notch interactions in endothelial cell activation. In xenograft models, they appear to target tumor angiogenesis by causing disorganized blood vessel development but do not have the same side effects caused by systemic Notch inhibitors. An additional potential advantage of biologics and mAbs in particular is the ability to conjugate them with radionuclides or toxins to selectively target cells that overexpress their targets. In the case of Notch pathway components, this approach would require a target that is at least relatively specific to cancer cells or significantly overexpressed by them (for example, Jagged-2 in multiple myeloma (Houde et al., 2004). The potential disadvantages of biologics in this setting include their generally complex dose–response curves in vivo and their long biological half-lives. If intermittent inhibition of Notch signaling is desirable to minimize adverse effects, using an mAb that will remain in circulation for days or weeks may prove challenging in terms of regimen design. Of course, the biological half-lives of mAbs can be modulated recombinant engineering or generation of F(ab)2s, F(ab)s or even single chain Fvs.

Indirect mechanisms of modulating Notch signaling without directly engaging pathway members with drugs or biologics can be envisioned. Modulating the expression of Notch receptors, ligands or downstream mediators is conceivable. This approach will require a detailed understanding of the transcriptional, translational and post-translational regulation of Notch pathway component expression in specific cellular contexts. At the moment, our knowledge in this area is still limited, with few exceptions. Expression of Jagged-1 was reported to be increased by nuclear factor (NF)-κB (Bash et al., 1999). Transcription of Notch-4 in endothelial cells is induced by AP-1 (Wu et al., 2005) and the glucocorticoid receptor (Wu and Bresnick, 2007), which uncharacteristically act synergistically at the Notch-4 promoter. Notch-1 has been reported to upregulate its own transcription (Deftos et al., 1998) and that of Notch-4 (Weijzen et al., 2002). The Ras (Weijzen et al., 2002), AKT (Liu et al., 2003) and mitogen-activated protein kinases (Zeng et al., 2005) pathways have been reported to stimulate Notch activity in some experimental models, suggesting that inhibitors of these pathways may affect Notch signaling in some situations. Some isoforms of transcriptional regulator Ikaros inhibit Notch-dependent transcription in normal and neoplastic T cells (Beverly and Capobianco, 2003; Bellavia et al., 2007; Kathrein et al., 2008). Corepressor SHARP (Oswald et al., 2002, 2005) associates with the Notch transcriptional complex and recruits CtIP/CtBP to block Notch-dependent transcription. If expression of Ikaros or SHARP could be modulated in specific cell types, this would result in selective Notch inhibition. In Drosophila, various microRNAs (miRNAs) regulate Notch signaling through the 3′-untranslated sequences of numerous Notch target genes (Kwon et al., 2005; Lai et al., 2005), and conversely miRNAs have been suggested to mediate some Notch effects in Caenorhabditis elegans (Yoo and Greenwald, 2005), raising the possibility that RNA-based therapeutics (siRNAs or miRNAs) could be used to modulate Notch signaling by targeting specific subsets of Notch targets. At the post-transcriptional level, Notch-1 protein levels and activity are regulated by several ubiquitin ligases that mediate either the degradation of Notch by polyubiquitination or conversely, its activation by monoubiqutination (Miele et al., 2006). One of these ligases (SEL10/Fbw7/Ago/hCDC4), responsible for the polyubiquitination and degradation of nuclear Notch-1 is mutated in some T-cell acute lymphoblastic leukemia (T-ALL) cases, resulting in prolonged Notch-1 signals (Malyukova et al., 2007; O'neil et al., 2007; Thompson et al., 2007). Desensitization of Notch-1 signaling by nonvisual β-arrestin and Kurz (Mukherjee et al., 2005) or endocytosis and degradation of Notch-1 mediated by Numb (Santolini et al., 2000; Pece et al., 2004) are also potential avenues to regulate Notch signaling if expression of Kurz or Numb can be modulated in cell-specific fashions.

Are there situations in which stimulation of Notch signaling can be envisioned as a therapeutic strategy in cancer? The strongest evidence for a tumor-suppressive role of Notch-1 is in epidermal keratinocytes (Nicolas et al., 2003; Koch and Radtke, 2007), because keratinocyte-targeted Notch-1 knockout in mice increases chemical carcinogenesis in the skin. However, it is difficult to envision chronic treatment of basal cell carcinomas with Notch ligands, given that these lesions can be generally cured by excision. In the context of tumor immunology, it is conceivable that Notch activation in T cells or other immunocytes may promote tumor rejection induced by a therapeutic tumor vaccine. This may be achieved, for example, by expressing a Notch ligand in antigen-presenting cells such as dendritic cells. We still do not have a clear understanding of the multiple functions of Notch signaling in the peripheral immune system, where it could modulate Th1-type (Minter et al., 2005) or Th-2-type (Tu et al., 2005) T-cell responses. It is conceivable that slightly different strategies for stimulation or slightly different signal strengths could produce either effect. Systemic delivery of a Notch stimulator may pose pharmacological challenges, because physiologically Notch ligands are transmembrane proteins. The mechanism of Notch activation requires trans-endocytosis of the Notch extracellular subunit NEC into the ligand-expressing cell, which in turn requires monoubiquitination of ligand intracellular tails (Miele et al., 2006). This is because the dissociation of NEC from the transmembrane subunit NTM requires significant mechanical strength to disrupt hydrophobic interactions in the NRR at the interaction site between the two subunits (Gordon et al., 2007). Thus, cell-associated or at least solid phase-bound ligands (for example, ligand-coated beads) are expected to be more effective than soluble ligands. Having said that, oligopeptides mimicking the conserved DSL region of Notch ligands can activate Notch receptors, albeit at high concentrations (Nickoloff et al., 2002). Fc fusion constructs that may associate with Fc receptors may be another strategy, particularly if the objective is immunomodulation.

Top

Toxicity

Although there is wide agreement that targeting cell-fate modulatory pathways is one of the most attractive new avenues in experimental cancer therapy, it would be naive to expect the modulation of such ancient, pervasively important pathways to have no adverse effects. Thus, key components of studying the possibility of targeting Notch in cancer must be the identification of clinically relevant toxicities and the development of strategies to prevent or ameliorate them.

Early clinical experience with GSIs indicates that the main adverse event in patients is dose-limiting secretory diarrhea caused by goblet cell metaplasia of the small intestine, which was first observed in preclinical models (Wong et al., 2004). The absence of myelotoxicity is welcome news in the setting of cancer chemotherapy. In mice, other adverse effects of systemic GSI treatment include reversible thymic suppression (Wong et al., 2004) and, in our hands, reversible hair depigmentation. Hair loss in dose-escalation experiments is an indication that a toxic dose has been reached and is associated with diarrhea and weight loss. Skin tumors have not been observed in humans and we have not observed them in mice treated for several weeks, though patches of mild, reversible hyperkeratosis are observed in nude mice. It is possible that life-long and/or complete shutdown of Notch signaling is required for more severe skin proliferative phenotypes. In both preclinical models and clinical studies, intermittent rather than continuous oral administration of GSIs greatly ameliorates the intestinal toxicity, presumably because it allows at least some intestinal stem cells to correctly differentiate as enterocytes. Parenteral administration of GSIs in mouse xenograft models in our hands was associated with less severe side effects, and doses that caused significant antineoplastic effects did not cause diarrhea or weight loss (Nickoloff et al., 2005), (Rizzo et al., submitted for publication). In mice, GSIs have immunosuppressive effects that may be undesirable under some circumstances. However, these effects may find clinical applications of their own in autoimmune disorders such as multiple sclerosis (Minter et al., 2005), and could potentially be indirectly useful in oncology in the treatment of graft-versus-host disease after bone marrow transplantation (Minter et al., 2005). GSIs are not significantly myelotoxic, making such a potential application at least theoretically feasible. Whether prolonged Notch inhibition by agents that pass the blood–brain barriers is associated with neurological toxicity is unknown at this time, but possible based on mouse data (Wang et al., 2004). At this time, it would appear that relatively short-term (days to weeks) systemic suppression of Notch signaling is consistent with a reasonable safety profile, especially in the context of cancer therapy. Perhaps the best way to use systemic Notch inhibitors in cancer may turn out to be as multiple cycles separated by ‘drug holidays’. Chronic, long-term suppression of Notch signaling is likely to require more selective agents, such as DLL4 antibodies in the context of antiangiogenic therapy, or more targeted delivery strategies of Notch inhibitors (for example, encapsulated within tumor-targeted nanocarriers).

Top

Looking to the future: rational combinations including Notch inhibitors and individualized medicine

Long-term therapeutic success in cancer is rarely achieved with monotherapy, and even targeting developmental pathways such as Notch will most likely require the development of combination regimens. Traditionally such regimens have been produced through a process of ‘clinical trial and error’, often based on limited mechanistic information. Clinical experimentation will always be necessary, because no preclinical model completely recapitulates a patient. However, the more complete and accurate our mechanistic understanding of how the pathways we target cross talk with each other, the less guesswork will be involved in designing future therapeutic regimens. The high evolutionary conservation of developmental pathways means that information from simpler model organisms is likely to be reliably predictive of human pathophysiology. On the other hand, the context dependence of Notch signaling will require each specific cancer type to be studied independently, without preconceived notions. Our knowledge is still considerably incomplete, but evidence accumulated so far suggests that some combination regimens involving Notch inhibitors deserve further investigation. The examples that follow are not meant to be all-inclusive.

Inhibitors of the PI3-kinase–AKT–mTOR pathway may be useful in combination with Notch inhibitors, and there is evidence that this strategy may reverse resistance to GSIs in T-ALL that carry PTEN inactivating mutation (Palomero et al., 2007). Whether this strategy can be successful in other cancers characterized by loss of PTEN is still unclear. The complex cross talk between Notch and NF-κB suggests that at least in some circumstances drugs that inhibit NF-κB activity directly or indirectly could be successfully combined with Notch inhibitors (Wang et al., 2006a, 2006b; Vilimas et al., 2007; Osipo et al., 2008). As intracellular Notch is degraded by the proteasome, and accumulates in cells treated with proteasome inhibitors, it is possible that these agents may benefit from the addition of a GSI. As DLL4 mAb appear to be effective independently of VEGF, they may be useful in combination with agents that block the VEGF pathway such as bevacizumab. Our data (Rizzo et al., submitted for publication; Osipo et al., submitted for publication) indicate that: (1) Notch signaling is prominently regulated by estrogen and Her2/Neu; (2) estrogen receptor-α (ERα)-negative and Her2/Neu-negative cancers have higher Notch activity and may respond to Notch inhibitors; (3) combinations of selective estrogen receptor modulators (SERMS) plus GSIs are particularly effective in ERα-positive tumors; (4) Combinations of trastuzumab plus GSIs or receptor tyrosine kinase inhibitors plus GSIs are synergistic in vitro and may be effective in Her2/Neu overexpressing, trastuzumab-resistant tumors. An unexpected but potentially useful observation was that co-treatment with tamoxifen greatly alleviated the intestinal side effects of orally administered GSIs, suggesting that this combination may be not only more effective but also safer than single agent GSI treatment.

To design the best combination regimens including Notch inhibitors, indication-specific studies will have to be performed. These studies will need to include a whole range of approaches, from simple model organisms such as Drosophila or zebrafish to in vitro and in vivo studies in mammalian models to identify which genetic and epigenetic factors interact with Notch signaling. These findings will require validation by studies of primary clinical specimens. Ultimately, the best use of these new therapeutic targets, as is the case for most new targeted agents, will be in the context of ‘individualized medicine’. It will be necessary to identify groups of patients and/or subtypes of cancer who are most likely to benefit from Notch inhibitors. To that end, we will have to determine: (1) which cancers and specific subtypes are characterized by active Notch signaling; (2) what role do specific components of Notch signaling perform in these cancers (for example, Notch-2 versus Notch-1), and whether global or selective Notch modulation is most desirable and (3) what genes or pathways cross talk with Notch in specific cancers, indicating targets for combination regimens. High-throughput system biology and bioinformatics will be important in this task. Simply determining expression levels of receptors and ligands in clinical specimens will not necessarily identify prospective responders. Because of extensive cross talk between Notch and other pathways, there is no simple correlation between expression and Notch activity. Thus, it will be important to develop accurate molecular tests that measure the level of pathway activity in vivo, possibly based on expression levels of multiple genes in the pathway. With the tools of today's cancer biology, these tasks are not as daunting as they would have been a few years ago. And the payoff for these efforts may be multiple new treatments for a whole range of human malignancies.

Top

References

  1. Allenspach EJ, Maillard I, Aster JC, Pear WS. (2002). Notch signaling in cancer. Cancer Biol Ther 1: 466–476. | PubMed | ISI |
  2. Bash J, Zong WX, Banga S, Rivera A, Ballard DW, Ron Y et al. (1999). Rel/NF-kappaB can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors. EMBO J 18: 2803–2811. | Article | PubMed | ISI | ChemPort |
  3. Bellavia D, Mecarozzi M, Campese AF, Grazioli P, Gulino A, Screpanti I. (2007). Notch and Ikaros: not only converging players in T cell leukemia. Cell Cycle 6: 2730–2734. | PubMed | ChemPort |
  4. Berman JN, Look AT. (2007). Targeting transcription factors in acute leukemia in children. Curr Drug Targets 8: 727–737. | Article | PubMed | ChemPort |
  5. Beverly LJ, Capobianco AJ. (2003). Perturbation of Ikaros isoform selection by MLV integration is a cooperative event in Notch(IC)-induced T cell leukemogenesis. Cancer Cell 3: 551–564. | Article | PubMed | ISI | ChemPort |
  6. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR et al. (2000). A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 5: 207–216. | Article | PubMed | ISI | ChemPort |
  7. Chen Y, De Marco MA, Graziani I, Gazdar AF, Strack PR, Miele L et al. (2007). Oxygen concentration determines the biological effects of NOTCH-1 signaling in adenocarcinoma of the lung. Cancer Res 67: 7954–7959. | Article | PubMed | ChemPort |
  8. Dallman MJ, Smith E, Benson RA, Lamb JR. (2005). Notch: control of lymphocyte differentiation in the periphery. Curr Opin Immunol 17: 259–266. | Article | PubMed | ISI | ChemPort |
  9. Deftos ML, He Y-W, Ojata EW, Bevan MJ. (1998). Correlating Notch signaling with thymocyte maturation. Immunity 9: 777–786. | Article | PubMed | ISI | ChemPort |
  10. Dickson BC, Mulligan AM, Zhang H, Lockwood G, O'Malley FP, Egan SE et al. (2007). High-level JAG1 mRNA and protein predict poor outcome in breast cancer. Mod Pathol 20: 685–693. | Article | PubMed | ChemPort |
  11. Fan X, Mikolaenko I, Elhassan I, Ni X, Wang Y, Ball D et al. (2004). Notch1 and Notch2 have opposite effects on embryonal brain tumor growth. Cancer Res 64: 7787–7793. | Article | PubMed | ISI | ChemPort |
  12. Farnie G, Brennan K, Clarke RB, Bundred NJ. (2005). Ductal Carcinoma in situ (DCIS) mammosphere formation effect of epidermal growth factor (EGF) and Notch signaling pathways on self-renewal capacity. Breast Cancer Res Treat 94(Suppl. 1): S14.
  13. Farnie G, Clarke RB. (2007). Mammary stem cells and breast cancer—role of Notch signalling. Stem Cell Rev 3: 169–175. | Article | PubMed | ChemPort |
  14. Farnie G, Clarke RB, Spence K, Pinnock N, Brennan K, Anderson NG et al. (2007). Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. J Natl Cancer Inst 99: 616–627. | Article | PubMed | ChemPort |
  15. Gordon WR, Vardar-Ulu D, Histen G, Sanchez-Irizarry C, Aster JC, Blacklow SC. (2007). Structural basis for autoinhibition of Notch. Nat Struct Mol Biol 14: 295–300. | Article | PubMed | ChemPort |
  16. Gupta-Rossi N, Six E, LeBail O, Logeat F, Chastagner P, Olry A et al. (2004). Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor. J Cell Biol 166: 73–83. | Article | PubMed | ISI | ChemPort |
  17. Hanahan D, Weinberg RA. (2000). The hallmarks of cancer. Cell 100: 57–70. | Article | PubMed | ISI | ChemPort |
  18. Houde C, Li Y, Song L, Barton K, Zhang Q, Godwin J et al. (2004). Over-expression of the NOTCH ligand JAG2 in malignant plasma cells from multiple myeloma patients and cell lines. Blood 104: 3697–3704. | Article | PubMed | ISI | ChemPort |
  19. Jundt F, Anagnostopoulos I, Forster R, Mathas S, Stein H, Dorken B. (2002). Activated Notch1 signaling promotes tumor cell proliferation and survival in Hodgkin and anaplastic large cell lymphoma. Blood 99: 3398–3403. | Article | PubMed | ISI | ChemPort |
  20. Jundt F, Probsting KS, Anagnostopoulos I, Muehlinghaus G, Chatterjee M, Mathas S et al. (2004). Jagged1-induced Notch signaling drives proliferation of multiple myeloma cells. Blood 103: 3511–3515. | Article | PubMed | ISI | ChemPort |
  21. Kathrein KL, Chari S, Winandy S. (2008). Ikaros directly represses the Notch target gene Hes1 in a leukemia T cell line: implications for CD4 regulation. J Biol Chem 283: 10476–10484. | Article | PubMed | ChemPort |
  22. Koch U, Radtke F. (2007). Notch and cancer: a double-edged sword. Cell Mol Life Sci 64: 2746–2762. | Article | PubMed | ChemPort |
  23. Kopan R, Ilagan MX. (2004). Gamma-secretase: proteasome of the membrane? Nat Rev Mol Cell Biol 5: 499–504. | Article | PubMed | ISI | ChemPort |
  24. Kukar T, Golde TE. (2008). Possible mechanisms of action of NSAIDs and related compounds that modulate gamma-secretase cleavage. Curr Top Med Chem 8: 47–53. | Article | PubMed | ChemPort |
  25. Kwon C, Han Z, Olson EN, Srivastava D. (2005). MicroRNA1 influences cardiac differentiation in Drosophila and regulates Notch signaling. Proc Natl Acad Sci USA 102: 18986–18991. | Article | PubMed | ChemPort |
  26. Lai EC, Tam B, Rubin GM. (2005). Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs. Genes Dev 19: 1067–1080. | Article | PubMed | ISI | ChemPort |
  27. Li K, Li Y, Wu W, Gordon WR, Chang DW, Lu M et al. (2008). Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of Notch3. J Biol Chem 283: 8046–8054. | Article | PubMed | ChemPort |
  28. Liu ZJ, Shirakawa T, Li Y, Soma A, Oka M, Dotto GP et al. (2003). Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis. Mol Cell Biol 23: 14–25. | Article | PubMed | ISI | ChemPort |
  29. Malyukova A, Dohda T, von der LN, Akhondi S, Corcoran M, Heyman M et al. (2007). The tumor suppressor gene hCDC4 is frequently mutated in human T-cell acute lymphoblastic leukemia with functional consequences for Notch signaling. Cancer Res 67: 5611–5616. | Article | PubMed | ISI | ChemPort |
  30. McKenzie GJ, Khan M, Briend E, Stallwood Y, Champion BR. (2005). Notch: a unique therapeutic target for immunomodulation. Expert Opin Ther Targets 9: 395–410. | Article | PubMed | ChemPort |
  31. Miele L. (2006). Notch signaling. Clin Cancer Res 12: 1074–1079. | Article | PubMed | ChemPort |
  32. Miele L, Golde T, Osborne B. (2006). Notch signaling in cancer. Curr Mol Med 6: 905–918. | Article | PubMed | ChemPort |
  33. Minato Y, Yasutomo K. (2005). Regulation of acquired immune system by Notch signaling. Int J Hematol 82: 302–306. | Article | PubMed | ChemPort |
  34. Minter LM, Turley DM, Das P, Shin HM, Joshi I, Lawlor RG et al. (2005). Inhibitors of gamma-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat Immunol 6: 680–688. | Article | PubMed | ISI | ChemPort |
  35. Mukherjee A, Veraksa A, Bauer A, Rosse C, Camonis J, Artavanis-Tsakonas S. (2005). Regulation of Notch signalling by non-visual beta-arrestin. Nat Cell Biol 7: 1191–1201. | Article | PubMed | ISI | ChemPort |
  36. Nam Y, Sliz P, Pear WS, Aster JC, Blacklow SC. (2007). Cooperative assembly of higher-order Notch complexes functions as a switch to induce transcription. Proc Natl Acad Sci USA 104: 2103–2108. | Article | PubMed | ChemPort |
  37. Nam Y, Sliz P, Song L, Aster JC, Blacklow SC. (2006). Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes. Cell 124: 973–983. | Article | PubMed | ChemPort |
  38. Nickoloff BJ, Hendrix MJ, Pollock PM, Trent JM, Miele L, Qin JZ. (2005). Notch and NOXA-related pathways in melanoma cells. J Investig Dermatol Symp Proc 10: 95–104. | Article | PubMed | ChemPort |
  39. Nickoloff BJ, Qin JZ, Chaturvedi V, Denning MF, Bonish B, Miele L. (2002). Jagged-1 mediated activation of Notch signaling induces complete maturation of human keratinocytes through NF-kappaB and PPARgamma. Cell Death Differ 9: 842–855. | Article | PubMed | ISI | ChemPort |
  40. Nicolas M, Wolfer A, Raj K, Kummer JA, Mill P, Van Noort M et al. (2003). Notch1 functions as a tumor suppressor in mouse skin. Nat Genet 33: 416–421. | Article | PubMed | ISI | ChemPort |
  41. Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S, Boland P, Gale NW et al. (2007). Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis. Novartis Found Symp 283: 106–120. | PubMed | ChemPort |
  42. O'neil J, Grim J, Strack P, Rao S, Tibbitts D, Winter C et al. (2007). FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to {gamma}-secretase inhibitors. J Exp Med 204: 1813–1824. | Article | PubMed | ISI | ChemPort |
  43. O'Neill CF, Urs S, Cinelli C, Lincoln A, Nadeau RJ, Leon R et al. (2007). Notch2 signaling induces apoptosis and inhibits human MDA-MB-231 xenograft growth. Am J Pathol 171: 1023–1036. | Article | PubMed | ChemPort |
  44. Okajima T, Irvine KD. (2002). Regulation of Notch signaling by O-linked fucose. Cell 111: 893–904. | Article | PubMed | ISI | ChemPort |
  45. Osipo C, Golde TE, Osborne BA, Miele LA. (2008). Off the beaten pathway: the complex cross talk between Notch and NF-kappaB. Lab Invest 88: 11–17. | Article | PubMed | ChemPort |
  46. Oswald F, Kostezka U, Astrahantseff K, Bourteele S, Dillinger K, Zechner U et al. (2002). SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. EMBO J 21: 5417–5426. | Article | PubMed | ISI | ChemPort |
  47. Oswald F, Winkler M, Cao Y, Astrahantseff K, Bourteele S, Knochel W et al. (2005). RBP-J{kappa}/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes. Mol Cell Biol 25: 10379–10390. | Article | PubMed | ChemPort |
  48. Palomero T, Sulis ML, Cortina M, Real PJ, Barnes K, Ciofani M et al. (2007). Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia. Nat Med 13: 1203–1210. | Article | PubMed | ChemPort |
  49. Parr C, Watkins G, Jiang WG. (2004). The possible correlation of Notch-1 and Notch-2 with clinical outcome and tumour clinicopathological parameters in human breast cancer. Int J Mol Med 14: 779–786. | PubMed | ISI | ChemPort |
  50. Pece S, Serresi M, Santolini E, Capra M, Hulleman E, Galimberti V et al. (2004). Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. J Cell Biol 167: 215–221. | Article | PubMed | ISI | ChemPort |
  51. Pinnix CC, Herlyn M. (2007). The many faces of Notch signaling in skin-derived cells. Pigment Cell Res 20: 458–465. | Article | PubMed | ChemPort |
  52. Pitsouli C, Delidakis C. (2005). The interplay between DSL proteins and ubiquitin ligases in Notch signaling. Development 132: 4041–4050. | Article | PubMed | ChemPort |
  53. Reedijk M, Odorcic S, Chang L, Zhang H, Miller N, McCready DR et al. (2005). High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res 65: 8530–8537. | Article | PubMed | ChemPort |
  54. Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC, Chanthery Y et al. (2006). Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature 444: 1083–1087. | Article | PubMed | ISI | ChemPort |
  55. Roy M, Pear WS, Aster JC. (2007). The multifaceted role of Notch in cancer. Curr Opin Genet Dev 17: 52–59. | Article | PubMed | ChemPort |
  56. Sansone P, Storci G, Giovannini C, Pandolfi S, Pianetti S, Taffurelli M et al. (2007). p66Shc/Notch-3 interplay controls self-renewal and hypoxia survival in human stem/progenitor cells of the mammary gland expanded in vitro as mammospheres. Stem Cells 25: 807–815. | Article | PubMed | ChemPort |
  57. Santolini E, Puri C, Salcini AE, Gagliani MC, Pelicci PG, Tacchetti C et al. (2000). Numb is an endocytic protein. J Cell Biol 151: 1345–1352. | Article | PubMed | ISI | ChemPort |
  58. Shih I, Wang TL. (2007). Notch signaling, gamma-secretase inhibitors, and cancer therapy. Cancer Res 67: 1879–1882. | Article | PubMed | ChemPort |
  59. Song LL, Miele L. (2007). Cancer stem cells—an old idea that's new again: implications for the diagnosis and treatment of breast cancer. Expert Opin Biol Ther 7: 431–438. | Article | PubMed | ChemPort |
  60. Thompson BJ, Buonamici S, Sulis ML, Palomero T, Vilimas T, Basso G et al. (2007). The SCFFBW7 ubiquitin ligase complex as a tumor suppressor in T cell leukemia. J Exp Med 204: 1825–1835. | Article | PubMed | ISI | ChemPort |
  61. Thurston G, Noguera-Troise I, Yancopoulos GD. (2007). The delta paradox: DLL4 blockade leads to more tumour vessels but less tumour growth. Nat Rev Cancer 7: 327–331. | Article | PubMed | ChemPort |
  62. Tu L, Fang TC, Artis D, Shestova O, Pross SE, Maillard I et al. (2005). Notch signaling is an important regulator of type 2 immunity. J Exp Med 202: 1037–1042. | Article | PubMed | ISI | ChemPort |
  63. Vilimas T, Mascarenhas J, Palomero T, Mandal M, Buonamici S, Meng F et al. (2007). Targeting the NF-kappaB signaling pathway in Notch1-induced T-cell leukemia. Nat Med 13: 70–77. | Article | PubMed | ISI | ChemPort |
  64. Wang Y, Chan SL, Miele L, Yao PJ, Mackes J, Ingram DK et al. (2004). Involvement of Notch signaling in hippocampal synaptic plasticity. Proc Natl Acad Sci USA 101: 9458–9462. | Article | PubMed | ChemPort |
  65. Wang Z, Banerjee S, Li Y, Rahman KM, Zhang Y, Sarkar FH. (2006a). Down-regulation of Notch-1 inhibits invasion by inactivation of nuclear factor-kappaB, vascular endothelial growth factor, and matrix metalloproteinase-9 in pancreatic cancer cells. Cancer Res 66: 2778–2784. | Article | PubMed | ISI | ChemPort |
  66. Wang Z, Zhang Y, Banerjee S, Li Y, Sarkar FH. (2006b). Inhibition of nuclear factor kappab activity by genistein is mediated via Notch-1 signaling pathway in pancreatic cancer cells. Int J Cancer 118: 1930–1936. | Article | PubMed | ChemPort |
  67. Weijzen S, Rizzo P, Braid M, Vaishnav R, Jonkheer SM, Zlobin A et al. (2002). Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nat Med 8: 979–986. | Article | PubMed | ISI | ChemPort |
  68. Wong GT, Manfra D, Poulet FM, Zhang Q, Josien H, Bara T et al. (2004). Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem 279: 12876–12882. | Article | PubMed | ISI | ChemPort |
  69. Wu J, Bresnick EH. (2007). Glucocorticoid and growth factor synergism requirement for Notch4 chromatin domain activation. Mol Cell Biol 27: 2411–2422. | Article | PubMed | ChemPort |
  70. Wu J, Iwata F, Grass JA, Osborne CS, Elnitski L, Fraser P et al. (2005). Molecular determinants of NOTCH4 transcription in vascular endothelium. Mol Cell Biol 25: 1458–1474. | Article | PubMed | ChemPort |
  71. Yan M, Plowman GD. (2007). Delta-like 4/Notch signaling and its therapeutic implications. Clin Cancer Res 13: 7243–7246. | Article | PubMed | ChemPort |
  72. Yoo AS, Greenwald I. (2005). LIN-12/Notch activation leads to microRNA-mediated down-regulation of Vav in C. elegans. Science 310: 1330–1333. | Article | PubMed | ChemPort |
  73. Zeng Q, Li S, Chepeha DB, Giordano TJ, Li J, Zhang H et al. (2005). Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling. Cancer Cell 8: 13–23. | Article | PubMed | ISI | ChemPort |
Top

Acknowledgements

This work was supported by NIH grant P01 AG2553101 and DOD IDEA grant W81XWH-04-1-0478. We are grateful to Antonio Pannuti for helpful discussions.

Top

MORE ARTICLES LIKE THIS

These links to content published by NPG are automatically generated

REVIEWS

The Notch ???gospel???

EMBO reports Review (01 Dec 2005)

See all 12 matches for Reviews

NEWS AND VIEWS

Notch in lymphopoiesis and T cell polarization

Nature Immunology News and Views (01 Jul 2005)

Promoting angiogenesis to a fault

Nature Biotechnology News and Views (01 Mar 2007)