The complex regulation of NF-ÎºB activation has presented significant challenges for the development of anticancer agents, but researchers are now beginning to better understand and embrace this complexity to drive development of a variety of novel NF-ÎºB-targeting strategies.
While anticancer therapies aimed at particular pathways have mushroomed in recent years, one crucial target has remained elusive: nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The pathway is constitutively active in the majority of cancers and provides a mechanistic link between chronic inflammation and tumorigenesis. As such, it represents an important target for anticancer therapy. The complex regulation of NF-κB activation has presented significant challenges for the development of such agents, but researchers are now beginning to better understand and embrace this complexity to drive development of a variety of novel NF-κB-targeting strategies.NF-κB was discovered in late 1980s as a nuclear factor that binds to the enhancer region of the kappa-light chain of immunoglobulin in B cells. It was subsequently shown to be a ubiquitous family of transcription factors that are present in all cells and control the transcription of more than 500 target genes involved in critical cellular pathways including proliferation and apoptosis. The NF-κB pathway is activated by a wide range of stimuli, including stress, cytokines, free radicals, ultraviolet irradiation, and bacterial or viral antigens.
In humans, there are five members of the NF-κB family: Rel-A, Rel-B, c-Rel, NF- κB-1, and NF-κB-2, which are able to form homo- and heterodimers with one another to produce a number of different NF-κB complexes with different activities. All family members share a Rel homology domain that is responsible for dimerization, nuclear translocation, DNA binding, and inhibition.
Under normal conditions, NF-κB is kept in an inactive state in the cytoplasm via the action of a family of inhibitors known as inhibitors of κB (IκBs), primarily IκBα. In response to an upstream stimulus, IκBα is phosphorylated by kinase enzymes, the IκB kinases (IKKs), and is subsequently targeted for degradation by the proteasomal pathway. Removal of IκBα inhibition releases the NF-κB complex, which is then free to migrate into the nucleus to initiate target gene expression.
About a decade ago it was discovered that there are in fact two NF-κB pathways with distinct mechanisms of regulation and nuclear targets. In the canonical pathway, which is induced by a variety of stimuli, NF-κB is activated primarily by IKKβ, while in the noncanonical pathway, which is induced by far fewer stimuli, NF-κB is activated primarily by IKKα, which in turn is activated by NF-κB-inducing kinase (IκBs). The two arms of NF-κB signaling have been shown to be equally important and are even interlinked in a number of ways.
EBV, indicates Epstein-Barr virus; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; HBV, hepatitis B virus; HCV, hepatitis C virus; HHV-8, human herpesvirus 8; HTLV, human T-lymphotropic virus; IL, interleukin; PDGFR, platelet-derived growth factor receptor; TNF, tumor necrosis factor.
Adapted from Sethi G, Sung B, Aggarwal BB. Nuclear factor-κB activation: from bench to bedside. Exp Biol Med. 2008; 233(1): 21-31.
NF-κB is activated by a number of upstream signaling pathways, including the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2), platelet-derived growth factor receptor (PDGFR), and c-KIT pathways. Another key activator of NF-κB is the receptor-activator of NF-κB (RANK), which is a type of tumor necrosis factor (TNF) receptor. Further adding to the complex regulation of NF-κB signaling, it undergoes numerous posttranslational modifications, including methylation and acetylation, and it interacts with several other transcription factors, such as signal transducer and activator of transcription (STAT)-3 and hypoxia inducible factor (HIF)-1α, both of which regulate NF-κB activity.
The activation of NF-κB plays a role in the suppression of apoptosis, stimulation of proliferation, and promotion of migration and invasion, all of which are hallmarks of cancer. Unsurprisingly, therefore, NF-κB has been implicated in tumorigenesis. What is surprising is that oncogenic mutations in the NF-κB genes are rare and largely limited to lymphoid malignancies, yet NF-κB activation is observed in almost all tumors.
Several potential explanations have been offered for this phenomenon. For example, NF-κB activation is in part driven by mutational activation of upstream signaling pathways, which are frequently mutated in cancer.
Recently, another explanation has come to light as researchers have discovered that NF-κB provides a crucial mechanistic link between cancer and chronic inflammation, such that oncogenic NF-κB activation may be the result of exposure to inflammatory signals in the microenvironment. Subsequently, activated NF-κB promotes the synthesis of inflammatory mediators (such as TNFα), which further enhances the malignant phenotype by stimulating cancer cell growth. Importantly, this means that therapeutic targeting of NF-κB could potentially provide anticancer activity both by directly targeting malignant cells and by targeting the inflammatory microenvironment that sustains tumorigenesis.
NF-κB also plays a role in the development of resistance to chemotherapy and radiation therapy; therefore, NF-κB-targeting agents have the potential to enhance the efficacy of these traditional therapies.Although NF-κB inarguably provides a promising target for anticancer therapy, the complexity of NF-κB regulation has made the development of agents challenging.
NF-κB can potentially be targeted at multiple points along its activation pathway, with key points being IKK activation, IκB degradation, and NF-κB DNA binding. Development efforts have focused particularly on inhibitors of IKKβ, to block the phosphorylation of IκBα, preventing its degradation and maintaining NF-κB in an inactive state in the cytoplasm. These agents include IMD-0354, BMS-345541, and PS-1145, but they are yet to proceed past the preclinical development stage.
In fact, there currently are no approved agents that directly target the NF-κB complex. However, many strategies for indirect targeting of NF-κB have been developed, and indeed many agents that were not originally conceived as NF-κB inhibitors have subsequently been shown to inhibit NF-κB, and these have yielded some significant advances (Table).
Mechanisms of Action
Status as Anticancer Agent
The proteasome is required to degrade the NF-κB inhibitor IκBα. These agents target the 26S proteasome and components of the ubiquitination machinery that target proteins for degradation by the proteasome.
FDA-approved for multiple myeloma (2003)
FDA-approved for multiple myeloma (2012)
Phase I/II trials in multiple myeloma (NCT01348919)
Phase I trials in multiple myeloma (NCT00461045) Phase I trials in advanced solid tumors or refractory lymphoma (NCT00629473, NCT00396864)
Phase I and I/II trials in patients with lymphoma and multiple melanoma (NCT00722488, NCT01415765)
Block phosphorylation and subsequent degradation of IκBα, either directly by binding components of the IKK kinase, or indirectly by inhibiting upstream signaling
Inhibits both canonical and noncanonical NF-κB pathways
PBS-1086 (Inhibits all Rel proteins)
Preclinical testing. Potent cytotoxicity demonstrated in multiple myeloma cell lines.
Inhibition of Upstream Signaling Components
Agents that target components of signaling pathways that are upstream of NF-κB have been shown to inhibit NF-κB activation
Denosumab (Xgeva) (RANK ligand inhibitor)
FDA-approved for prevention of skeletal-related events in patients with bone metastases from solid tumors (2010)
NF-κB undergoes numerous types of posttranslational modification, including acetylation, which regulate its activity.
FDA-approved for cutaneous T-cell lymphoma (2006)
FDA-approved for cutaneous T-cell lymphoma (2009)
Preclinical testing. Recent report of discovery of thieno[3,2-d]pyrimidine- 6-carboxamides as potent inhibitors of SIRT1, 2, and 3a
Gene transfer of inhibitory proteins of NF-κB activation
Nonphosphorylatable form of IκBα that cannot be degraded and therefore prevents activation of NF-κB
Preclinical testing. A number of trials have shown that administration of a nonphosphorylatable mutant of IκBα with anticancer agents increases the chemosensitivity of cancer cells.
Cell-Permeable Peptide Inhibitors
Small peptides that inhibit NF-κB activation
SN-50 (consists of the nuclear localization sequence of p50, which inhibits the nuclear transport machinery, thereby blocking the ability of NF-κB to enter the nucleus)
Preclinical testing. Substantially sensitizes the anticancer activity of cisplatin in ovarian cancer cells
NEMO-binding domain peptide (inhibits the IKK complex that activates NF-κB)
Antisense oligonucleotides targeted against NF-κB genes, interfering with their expression
Rel-A antisense oligonucleotides
Preclinical testing. Pronounced inhibition of tumorigenesis in murine models
A number of natural compounds have been shown to inhibit NF-κB activation.
Curcumin (a polyphenol from the plant Curcuma longa, found in turmeric)
Phase II trials in advanced pancreatic cancer (NCT00094445) and breast cancer (NCT01740323), among others
Resveratrol (naturally occurring polyphenol found in grapes, peanuts, and wine)
Completed phase I studies in colon cancer (NCT00256334)
Parthenolide (a sesquiterpene lactone found in the herbal medicine feverfew)
aDisch, et al. J. Med Chem. 2013;56(9):3666-3679
Of particular note are proteasome inhibitors. These agents exploit the fact that IκBα is degraded by the proteasomal degradation pathway; thus, inhibiting proteasome activity indirectly inhibits NF-κB activation by preventing IκBα degradation. Bortezomib (Velcade) became the first FDA-approved proteasome inhibitor in 2003 and was a major breakthrough for the treatment of multiple myeloma, conveying a significant survival benefit in combination with prednisone and melphalan in patients with previously untreated disease and when compared with dexamethasone in patients with relapsed disease.
Second-generation proteasome inhibitors act at a lower concentration with lower toxicity and can be administered orally. These include carfilzomib (Kyprolis), which was recently approved for the treatment of patients with multiple myeloma who have received at least two prior therapies, including bortezomib, following clinical trials in which nearly one-quarter of patients experienced complete or partial disappearance of their tumors with a median duration of response of 7.8 months.
Other strategies to target NF-κB activation include using inhibitors of the upstream signaling pathways mentioned above. Many of these were not conceived as NF-κB inhibitors but have subsequently shown NF-κB inhibitory activity. Among them is an antibody against the ligand that binds to RANK, one of the main activators of NF-κB signaling. Denosumab (Xgeva) has been approved since 2010 for the prevention of skeletal-related events in patients with bone metastases from solid tumors.
NF-κB undergoes numerous types of posttranslational modification, including acetylation, which regulates its activity. The histone deacetylase enzyme HDAC3 acts directly on Rel-A to enable its association with IκBα, and the sirtuins are a family of deacetylase enzymes, with several members that act on NF-κB. Thus, HDAC inhibitors provide a potential therapeutic means of indirectly inhibiting NFκB. Vorinostat (Zolinza) and romidepsin (Istodax) are two such agents, both approved for the treatment of cutaneous T-cell lymphoma. Sirtuin inhibitors have also drawn a significant amount of attention and are currently in preclinical development.
Nonsteroidal anti-inflammatory drugs such as ibuprofen, aspirin, and indomethacin have been shown to suppress NF-κB activation, though their exact mechanism of action is not fully understood. A number of natural products with anti-inflammatory properties are also potent inhibitors of NF-κB. These include the polyphenolic compounds curcumin and resveratrol, which are undergoing phase II and phase I clinical testing, respectively, in a number of different cancer types.
Finally, since the noncanonical NF-κB pathway was discovered, it has been increasingly recognized as an important arm of NF-κB signaling. The activity of IKKβ inhibitors has been shown to be somewhat limited in vivo; this could be because these inhibitors only target the canonical pathway, and there may be compensatory activity by the noncanonical pathway. With this in mind, a dual inhibitor of both pathways is being developed, which targets all five members of the NF-κB gene family. PBS-1086 is currently being examined in preclinical studies and has demonstrated potent cytotoxicity in multiple myeloma cell lines.
Jane de Lartigue, PhD, is a freelance medical writer and editor based in Davis, California.