11001_The Role Of Traf3 And Cyld Mutationin The Etiology Of Human Papillomavirus Driven Head And Neck Cancers

B was not restricted to B-cells but rather ubiquitously observed in all cells, hinting at an
ancient and conserved origin for this protein.38 Second, a series of experiments by Patrick
Baeuerle and Baltimore led to the discovery that DNA binding by NF-κB following stim-
ulation with lipopolysaccharide (LPS) did not require ”de novo protein synthesis”, hinting
that this factor was present but inactive under normal conditions. When NF-κB binding
of DNA was shown to increase following the application of translational inhibitors, it was
hypothesized that the agent responsible for inactivating NF-κB was transient. In 1988, the
family of agents responsible was identified and named inhibitor of kappa-b (IkB).39
A Nature review commemorating 25 years since the discovery of NF-κB stated that over
35,000 articles have been written about NF-κB and its biology, signaling pathways, and
effects of the immune system, response to infection, and cell survival. Here, we present a
brief overview of NF-κB as it relates to our work in head and neck cancer.38 While it was
clear that NF-κB was broadly consequential to immunology, the discovery that one NF-
B subunit called p50 had homology to the avian oncoprotein v-Rel presaged NF-κB’s
relevance in pathways of carcinogenesis as well.39
Today, we understand that the transcription factor NF-κB is actually a family of five re-
lated proteins that share the Rel domain, which is responsible for dimerization and DNA
INTRODUCTION
13
binding. These proteins are: p65 (relA), c-Rel, RelB, p105 and p100. p105 and p100 are
precursor proteins that are degraded to p50 and p52, respectively. Prior to activation and
DNA binding, these subunits dimerize. The most common combinations are p50-p65
(RelA) and p52-RelB. Activation of NF-κB to the point where these two heterodimers
can bind DNA and execute their downstream effects is the result of two respective, tightly-
regulated pathways.40
In both pathways, the penultimate step to activation is release of the NF-κB het-
erodimers by the inhibitory factor IkB. Cytoplasmic sequestration of NFkB by IkB is over-
come when IkB is phosphorylated by IkB Kinase or IKK. IKK itself is a family of proteins:
two catalytically active subunits IKKa and IKKb and a regulatory subunit IKKgamma (also
called NEMO). The phosphorylation of NEMO regulates whether IKK is active and able
to phosphorylate IkB.40
The canonical pathway is more commonly activated by physiologic stimuli. In this path-
way, cytokine signals from receptors such as tumor necrosis factor receptor (TNFR) and
interleukin 1 (IL-1) receptor (IL-1R), as well as markers of infection such as Toll-like re-
ceptor 4 (TLR4) lead IKKb and NEMO to phosphorylate IkB and lead to the transloca-
tion of p50-p65 heterodimers into the nucleus. Conversely, the non-canonical pathway
is primarily activated by factors such as CD40 ligand, BAFF
, and lymphotoxin-2. In this
pathway, IKKa phosphorylation mediates processing of p100-RelB complexes into p52-
RelB complexes which are active and can translocate into the nucleus. This degradative
processing is activated by NF-κB inducing kinase (NIK). At baseline, the p100 subunit
plays an inhibitory role until it is processed into p52. Interestingly, while canonical ac-
tivation of NF-κB is rapid, non-canonical signaling is slower and occurs on the order of
hours3.41
Given the highly conserved nature of this regulatory pathway, most regulation and mod-
ification to NF-κB biology is seen between the initial receptor-mediated signaling that
occurs at the cell membrane and the activation of the IKK complex. This regulation relies
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14
Figure 3. The Role of TRAF3 and CYLD in the NF-κB pathway. TRAF3 is an E3 ubiquitin ligase
that adds ubiquitin moieties to NIK, marking it for proteasomal degradation. Once degraded, NIK
can no longer activate IkKa, which activates NF-κB in the non-canonical pathway. CYLD is a deu-
biquinating enzyme that removes ubiquitin moieties from NEMO, the regulatory subunit of IKK.
By doing so, it prevents IKK from phosphorylating IkB and releasing active NF-κB in the canonical
pathway. In this figure, IKKa, IKKb, and NEMO are shown separately for simplicity. In reality, these
three proteins form the IKK complex. Thus, TRAF3 and CYLD serve as negative regulators of NF-
κB signaling. Absense or inactivation of either of these two proteins leads to constitutive activation
of NF-κB. While CYLD primarily acts in the canonical pathway and TRAF3 primarily acts in the
non-canonical pathway, significant crosstalk between the two pathways suggests that this demarca-
tion may not be absolute.
INTRODUCTION
15
heavily on ubiquitin-dependent signaling.
The ubiquitin system is responsible for the degradation of proteins, allowing for the re-
cycling of amino acids for other biochemical processes and regulating the turnover of pro-
teins whose expression is required transiently. Ubiquitin itself is a protein of modest size
comprising of only 76 amino acids. Ubiquitin is added to proteins as a molecular tag via
a three-step pathway. First, ubiquitin is activated by the protein E1. Second, the carrier
protein E2 binds ubiquitin. Finally, an E3 ubiquitin ligase binds the protein of interest and
catalyzes the addition of ubiquitin to a Lysine residue of that protein. The rich diversity of
E3 ubiquitin ligases and their specificity towards specific substrate proteins allows for pre-
cise targeting and regulation in the ubiquitin pathway.42,43 This process is repetitive and
results in the synthesis of poly-ubiquitin chains on proteins. In addition, there are times
when removal of ubiquitin is required for appropriate signaling. For this purpose, proteins
called deubiquitinases catalyze the hydrolysis of ubiquitin from a poly-chain.43
Ubiquitination typically occurs on lysine residues, either on the substrate proteins or
on other ubiquitin proteins themselves in order to form chains. The nature of this lysine
bond, however, can drive the ultimate fate of ubiquitin-mediated signaling. Ubiquitin it-
self has seven lysine residues, of which the most commonly used are K48 and K63. K48
linked poly-ubiquitin tails mark proteins for degradation by the 26S proteasome. In NF-
κB signaling, IkB is degraded by a K48-linked poly-ubiquitination mechanism. On the
other hand, K63 linked poly-ubiquitin tails play a role in non-proteolytic signaling. Deu-
biquinating enzymes (DUB) play an especially important role in the latter type of signal-
ing.42,43,44
Among the proteins that utilize ubiquitin to drive downstream signaling, the TNFR-
associated factor (TRAF) family of proteins appears to play a critical role. TRAFs assemble
into protein complexes on the intracellular surface of membranes where they can relay sig-
nals from membrane receptors to downstream effectors.40 In this role, they are responsible
for regulating signaling in various immunologic, inflammatory, and cell survival pathways
INTRODUCTION
16
as well as crosstalk between pathways. In total, seven TRAF proteins (TRAF1-7) have
been described. All of them share a TRAF domain that facilitates binding to surface re-
ceptors and creating protein complexes that enable downstream signaling. Interestingly, all
except TRAF1 appear to have E3 ubiquitin ligase capability, highlighting both their struc-
tural and functional role in pathway regulation.40 In canonical NF-κB signaling, TRAF2
associates with two E3 ubiquitin ligases called central Inhibitor of Apoptosis (cIAP) 1 and
2 as well as the kinase RIP1. K63-linked polyubiquitination of RIP1 by cIAP1 and cIAP2
leads to activation of IKK and activation of NF-κB signaling. In the non-canonical path-
way, TRAF3, an E3 ubiquitin ligase, drives polyubiquitination of NIK and leads to its
degradation, preventing processing of p100-Rel by IKKa. Thus, TRAF3 exhibits an in-
hibitory effect on NF-κB signaling. Interestingly, TRAF2 and cIAP2 also degrade TRAF3
so that TRAF3 depletion and subsequent NF-κB signaling is transient.40,45,46 This is one
example of cross-talk between the canonical and non-canonical pathways.40 NIK and
non-canonical NF-κB activity was shown to be elevated in multiple myeloma, the result
of NIK amplification or deletion of TRAF2, TRAF3, cIAP1, and cIAP2.45,16
While the role of E3 ubiquitin ligases in NF-κB mediated signaling has been clearly es-
tablished, the discovery of CYLD as a deubiquitinating enzyme heralded the discovery that
removing ubiquitin from proteins was also a key part of this pathway. Located on chromo-
some 16, the Cylindromatosis (CYLD) gene encodes a 956 amino acid protein including
a C-terminal domain that high conserved among deubiquitinating enzymes. CYLD is a
tumor suppressor gene that was found to be mutated in familiar cylindromatosis, a con-
dition marked by numerous benign tumors of the skin appendages; however, it’s function
remained unclear until its discovery as a deubiquitinating enzyme.47,48,49
Brummelkamp and colleagues identified 50 candidate deubiquitinating genes contain-
ing the aforementioned C-terminal domain common to all deubiquitinases. They gener-
ated small-hairpin RNAs against each of these candidate genes to knock-down expression
and then tested NF-κB activity in U2-OS cells using a luciferase reporter assay. The only
INTRODUCTION
17
gene that was found to be associated with NF-κB activation was CYLD.50 Subsequent
molecular studies identified that CYLD bound to NEMO, TRAF2, and TRAF6. CYLD
is thought to hydrolyze K63-linked ubiquitin chains on target proteins. In canonical NF-
B signaling, deubiquitination of NEMO prevents it from activating IkB, thereby abro-
gating activation and translocation of p52-p60.48,49 CYLD association with TRAF2 and
TRAF6 also suggest a role in the non-canonical pathway. In fact, CYLD deletions were
also associated with multiple myeloma in the previously mentioned study.16
In summary, TRAF3 inhibits NF-κB signaling through the non-canonical pathway
while CYLD inhibits NF-κB signaling through the canonical pathway. However, due
to cross-talk between both pathways, some crossover in this mechanism is not precluded.
Overall, inactivating mutations in TRAF3 or CYLD disinhibits NF-κB signaling, resulting
in constitutive translocation of active NF-κB into the nucleus and persistent expression of
NF-κB induced gene.
Previous Work
While TRAF3, CYLD, and other genes of this pathway had known functions in NF- ]>