Molecular targets for novel drug development in pancreatic cancer Review

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ISSN 2058-7775
Review
Molecular targets for novel drug development in
pancreatic cancer
Eimear O’ Reilly1, Corrado Santocanale1 and Eva Szegezdi2*
1
Centre for Chromosome Biology and National Centre for Biomedical Engineering Sciences, Ireland, 2Apoptosis Research
Centre, School of Natural Sciences, National University of Ireland, Galway, Ireland.
*Corresponding author:
Eva Szegezdi
[email protected]
Editors approved:
Paul J. Higgins
Albany Medical College, USA
Vicente Notario
Georgetown University Medical
Center, USA
Lan Mo
Weill Cornell Medical College, USA
Sucharita SenBanerjee
Harvard Medical School, USA
Received: 20 January 2015
Revised: 05 March 2015
Accepted: 27 March 2015
Published: 06 April 2015
Abstract
Pancreatic Ductal Adenocarcinoma (PDAC), more commonly known as pancreatic
cancer, is one of the primary contributors to cancer-associated death in western
countries. Despite constant effort and medical developments, conventional treatments
such as chemotherapy, surgery and radiation therapy have proven to be ineffective
in curing PDAC. These forms of treatment only provide palliative relief. Most PDAC
patients develop metastasis, resulting in eventual death. The underlying cause of this
cancer remains poorly understood. However, genetic alterations, including mutations
of oncogenic KRAS, NF-κB pathways, and tumour suppressor genes have recently
been associated with PDAC formation. Novel therapeutic strategies targeting these
mutations are being developed and tested, some of which are exhibiting promising
results. Targeting the oncogenic mutations may have the potential to revert the
aggressive nature of this cancer, providing a new window opportunity for the battle
against pancreatic cancer. In this review, we summarise the aberrant molecular
pathways associated with PDAC development and progression and the current status
of treatment strategies aiming to block these pathways.
Keywords: Pancreatic cancer, oncogenic mutation, molecular targets, NF-κB, KRAS,
targeted therapy
Introduction
© 2015 Szegezdi et al; licensee
Vernon Innovative Publishers.
http://creativecommons.org/
licenses/by/4.0.
T
he pancreas is a secretory organ containing both endocrine and exocrine glands. The exocrine
glands, which account for 95% of the glands, produce the pancreatic juice containing the digestive
enzymes. The endocrine cells are present in the islets of Langerhans. These islets are responsible for
the production of the two main metabolic hormones, insulin and glucagon [1]. Pancreatic cancer is
the tenth most common cancer representing 2.8% of cancer cases in the USA [2] and 3% in the UK,
equally affecting males and females [3]. The most prevalent form of pancreatic cancer is Pancreatic
Ductal Adenocarcinoma (PDAC), the malignant transformation of the exocrine duct cells [4]. PDAC,
constituting 90% of all pancreatic cancer cases, displays local invasion and distant metastasis during
early disease stages. It is a very aggressive cancer type with an overall 5-year survival of only 6%
[5,6]. In 2014, 46,420 new cases and 39,590 deaths were recorded in the USA [2], while the annual
incidence of PDAC in the UK is approximately 8,700 cases [3]. A major cause of the poor prognosis
of PDAC is the frequent metastasis of PDAC cells to the liver, lungs, and other organs.
Unfortunately, as it is also the case with many other cancer types, the symptoms of pancreatic
are generally non-specific. These include abdominal pain, unexplained weight loss, diarrhoea,
constipation, bloating, vomiting, jaundice and malaise [7]. Tumour growth can result in bile duct
blockage, which may lead to a build-up of bilirubin, causing jaundice. Tumours can also block the
duodenum, causing nausea, vomiting and constipation [8].
To date, surgery is the only chance of cure. Unfortunately, due to the highly non-specific symptoms,
at the time of diagnosis the vast majority of patients already have advanced disease and are not
How to cite this article:
O’ Reilly, E., Santocanale, C. and Szegezdi, E. (2015). Molecular targets for novel drug development in pancreatic cancer. Annals of Cancer
Research, 2:3. Retrieved from http://www.vipoa.org/cancer
Annals of Cancer Research
suited for surgical resection [4]. Even in patients who undergo However, a clear link has been established between excessive
resection, 80% eventually relapse and succumb to the disease alcohol consumption, chronic pancreatitis and development of
[9,10]. Systemic therapies are largely ineffective, increasing the pancreatic cancer as a number of large-scale studies linked chronic
overall survival by only a few months on average. Consequently, pancreatitis with an increased risk of pancreatic cancer [15].
there is an urgent need to develop a better understanding of the
Obesity is another underlying risk factor. Individuals suffering
molecular background of pancreatic cancer, its heterogeneity from obesity have exhibited a 20% greater chance of developing
and how this may relate to chemotherapy responses.
PDAC [16,17]. Further supporting the potential contribution of
metabolic disorders to PDAC development, diabetes has been
Review
identified as another risk factor [18]. However, the causative
Risks factors of pancreatic ductal adenocarcinoma link between diabetes and pancreatic cancer remains unclear
The causes of pancreatic cancer remain largely undetermined. as development of PDAC itself can lead to diminished insulin
However, environmental factors are believed to be important production and subsequent diabetes. In patients with diabetes
contributors to the development of pancreatic cancer (Table 1). mellitus (DM), the chance of developing PDAC is increased by
Tobacco emerged as a prevalent risk factor in PDAC development. 40-100% [19]. It has been reported that the majority of PDAC
Cigarette smokers have an up to 3.6 times greater chance of (80-90%) patients have type II diabetes [20], although studies
developing pancreatic cancer [11] and recent figures show that have shown a two-fold increased risk of pancreatic cancer in
smoking is accountable for 29% of all PDAC cases in the UK [12]. patients with type I diabetes as well [20]. While environmental
It is believed that carcinogens released from the tobacco smoke factors seem to play a central role in PDAC development, even
are responsible. Due to the fact that only a small proportion of possibly to a higher extent than in case of some other cancers,
smokers develop PDAC, the assumption is that some individuals the mechanism how they trigger or potentiate the malignant
are more susceptible due to a less effective protective system transformation is not well understood.
against the toxic/carcinogenic molecules in the tobacco smoke.
Studies are beginning to identify familial links in pancreatic
For example, it is believed that mutations or polymorphisms cancer. For example, Permuth-Wey and Egan have shown that
in enzymes of the cytochrome P system (e.g., CYP1A2) and there is almost a 2-fold increased risk in developing PDAC when
n-Acetyl transferase enzymes affects their activity and ability there is a family history of the disease [21]. Another familial
to detoxify tobacco products and may be an underlying cause linkage associated with the onset of pancreatic cancer is inherited
of this susceptibility [13].
pancreatitis, a rare autosomal disease. Its symptoms are similar
to those of chronic pancreatitis [22,23]. Other germ-line diseases
have also been linked to small subsets of PDAC, the best known
Table 1. Epidemiology of pancreatic cancer: 1970’s versus current
data. Table adopted from [61].
of these are listed in Table 2.
1975 data
Current data
Common in
western countries
Still true [5].
Current treatment methods
Increased risk for
smokers
Confirmed [12,61].
Surgeons would consider tumours of an exocrine origin to
be ‘resectable’ if the cancer has not metastasized beyond the
pancreas [7]. Resectable tumours are usually removed by the
‘Whipple’ procedure (pancreaticoduodenectomy) or by a distal
Increased risk for
diabetics
Type II and type I diabetes increases
risk [20].
Table 2. Diseases associated with the onset of PDAC. Table
adopted from [61].
Alcohol
consumption
No clear correlation.
Obesity
20% increased risk of developing pancreatic
cancer [16].
Hereditary
pancreatitis
High risk of pancreas cancer in patients
with hereditary pancreatitis [15,22,23].
Increasing frequency Yes, in both males and females [3].
Alcohol consumption is another proposed risk factor. Studies
revealed that drinking three or more alcohol beverages each
day increases the chance of developing pancreatic cancer by
20%. This has been linked to pancreatitis induced by alcohol
consumption [14]. In today’s culture, a strong relationship exists
between alcohol consumption and cigarette smoking making
it difficult to determine the individual contribution of alcohol
consumption versus tobacco smoke to PDAC development [14].
O’ Reilly et al. 2015
Disease
Chromosome Comments
Li-Fraumeni
syndrome
17p13.1
Defective p53. Modest
increased risk of
pancreatic cancer [62].
Cystic Fibrosis
7q31
Increased risk of
pancreatic tumours [63].
Peutz-Jeghers
syndrome
19p
Mutation may
contribute to both
sporadic and inherited
disease [64].
Familial adenomatous
polyposis
5q12–21
Mutation found in
pancreas cancers [64].
Hereditary
non-polyposis colon
cancer (HNPCC)
2, 3
Risk of developing
pancreatic cancer.
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Annals of Cancer Research
pancreatectomy [7]. Staging of PDAC is however a difficult task,
which may explain why the cancer may return after surgery.
As a result chemotherapy drugs, such as gemcitabine (Table 3),
are used as an adjuvant therapy with the aim to eradicate
potential disseminated tumour cells [24]. Unfortunately, the
majority of patients receive little or no benefit from current
chemotherapies mainly because most of the cancer cells are
either intrinsically chemoresistant or they become resistant
during therapy. In addition, extrinsic mechanisms such as the
tumour stroma have been discussed as a driver of resistance to
chemotherapy in PDAC [25].
Therefore, it is of great interest to identify and characterize
what molecules promote drug resistance with the hope to inhibit
these molecules as novel targeted therapy options.
Locally advanced pancreatic cancers are difficult to treat
with surgery. The combination of chemotherapy and radiation
(chemoradiation) is often used to help reduce the tumour size [7].
However, this is an intensive form of treatment; the combination
could result in severe side effects for the patient. Metastatic PDAC
is also difficult to tackle with surgery. Chemotherapy drugs such as
gemcitabine, albumin-bound paclitaxel nanoparticles (Abraxane)
or the combination of four chemotherapy drugs, known as
FOLFIRINOX (Folinic Acid, Fluorouracil, Irinotecan Hydrochloride,
Oxaliplatin) are the standards of care for metastatic disease [24]
(Table 3). The intensity of this treatment, once again, can result
in severe side effects for the patients, including fever, diarrhoea,
vomiting, exhaustion and neutropenia [24].
Recent findings in the US have shown that by reducing the
administration of Abraxane to every other week, the side effects
of the treatments could be reduced without compromising
the efficacy of the treatment [26]. However, the side effects of
chemotherapy are still significant. Moreover, these treatments
have limited efficacy, often only improving the quality of life
of patients for a period of time rather than curing the disease
itself. Thus, understanding the molecular drivers of PDAC
progression is crucial as this would allow identification of
alternative treatment strategies.
Molecular alterations in pancreatic cancer emerge
as novel therapeutic targets
Recent advancements have determined that there are specific
genetic alterations associated with the development of
Table 3. Current available treatments for pancreatic cancer [65-67].
Treatment
Comments
Surgery
Procedure to remove pancreatic tumours.
• Whipple procedure.
• Total pancreatectomy.
• Distal pancreatectomy.
Types of palliative surgery to remove symptoms and improve quality of life.
• Surgical billary bypass.
• Endoscopic stent placement.
• Gastric bypass.
Radiation therapy
Chemotherapy
Combinational chemotherapy
Chemoradiation
O’ Reilly et al. 2015
•
•
External radiation.
Internal radiation utilised sealed radioactive substances in close proximity to the tumour.
Food and Drug Administration (FDA) approved drugs.
• Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation).
• Adrucil (Fluorouracil).
• Afinitor (Everolimus).
• Efudex (Fluorouracil).
• Erlotinib Hydrochloride.
• Everolimus.
• Fluoroplex (Fluorouracil).
• Fluorouracil.
• Gemcitabine Hydrochloride.
• Gemzar (Gemcitabine Hydrochloride).
• Mitomycin C.
• Mitozytrex (Mitomycin C).
• Mutamycin (Mitomycin C).
• Paclitaxel Albumin-stabilized Nanoparticle Formulation.
• Sunitinib Malate.
• Sutent (Sunitinib Malate).
• Tarceva (Erlotinib Hydrochloride).
•
•
•
•
FOLFIRINOX (Folinic Acid, Fluorouracil, Irinotecan Hydrochloride, Oxaliplatin).
GEMCITABINE-CISPLATIN.
GEMCITABINE-OXALIPLATIN.
OFF (Oxaliplatin, Fluorouracil, Folinic Acid).
Combinations of chemotherapy and radiation therapy to increase the effects of both.
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Annals of Cancer Research
tumorigenesis and pathogenesis of pancreatic cancer. These
include activation of oncogenes, inactivation of tumour suppressor
genes and overexpression/overactivation of proliferation
pathway drivers. The discovery of the genetic alterations in
PDAC provides a window of opportunity for the identification
of molecular targets for treatment. Due to the central role of
NF-κB and KRAS in driving pancreatic cancer, the following
section discusses the feasibility of targeting these molecules and
the current status of research.
Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of
the best characterised oncogenes as a key mediator of growth
factor signal transduction [27]. Oncogenic mutations of KRAS
are the most common mutations detected in PDAC [28]. The
KRAS protein is a GTPase protein, acting as molecular switch
for RAF activation and consequent activation of the mitogen
activated protein kinase cascade inducing cell division [28].
Oncogenic mutation of KRAS usually results in a single
amino acid substitution that leads to reduced GTPase activity,
extending the time period RAS stays active after stimulation,
affecting mitogen activated protein kinase (MAPK) signalling
and consequent uncontrolled growth of cells [28].
Mutation of glycine at position 12 to aspartate (KRASG12D)
is the most common KRAS mutation in pancreatic cancer [28].
These mutations are primary events in PDAC, with mutated
KRAS is detected in 90% of low-grade pancreatic intraepithelial
neoplasia lesions (PanIN) [29].
Interestingly, when tissues from healthy individuals and
cancer patients were compared, oncogenic KRAS was detected
in a significant number of healthy tissues, including the pancreas,
colon and lungs [28]. Additionally, only a small fraction mutated
KRAS-expressing cells of KRASG12D-expressing mice develop
cancers, implying that KRAS alone is not sufficient to initiate
carcinogenesis and other co-operating genetic or epigenetic
factors that lead to elevated KRAS activity are required launch
fully fetched malignant transformation in KRAS mutated cells.
Studies that support this hypothesis have shown that upstream
KRAS signals, such as epidermal growth factor and inflammatory
stimuli, may be critical for carcinogenesis [30].
(UPP) supplies an alternative method of KRAS targeting for pancreatic cancer. The UPP is composed of the proteasome complex
and a ubiquitinating enzyme family [33]. These include the
ubiquitin-activating enzymes (E1), the ubiquitin-conjugating
enzymes (E2) and the ubiquitin ligases (E3). E3 is the enzyme
that recognises the target substrate, in this case KRAS, and
tags it with a poly-ubiquitin tail where the ubiquitin units
are connected after each other through their lysine 48 (K48)
residue [33]. The work by Ma and colleagues helped to develop
an engineered U-box based chimeric E3 ligase, known as RC-U,
which is able to trigger the degradation of KRAS through the
UPP. The specificity of the ubiquitin ligase (E3) is a key factor in
targeting the KRAS oncogene. RAF1 (v-raf-1 murine leukaemia
viral oncogene homolog 1) is an essential downstream effector
of RAS, specifically activated by KRAS [34]. Studies have shown
that the RAS-binding domain (RBD) and the cysteine-rich
domain (CRD) of RAF1 interacts with KRAS in vivo [34] and
thus were chosen as the interaction domain of the engineered
E3 ligase. The E3 ligase, used by Ma and colleagues, had a
RBD+CRDRAF1-U-Box (RC-U) structure [34].
It was shown that ectopic expression of RC-U in mutated
KRAS expressing PANC-1 cells and MIA PaCa-2 cells reduced
the levels of KRAS protein [34]. To confirm if the decreased
level of KRAS protein was a result of the presence of the RC-U
E3 ligase, an ubiquitination assay was conducted. The test
established that KRAS was in fact polyubiquitinated in the
presence of RC-U [34]. Moreover, ectopic expression of RC-U
significantly reduced PANC-1 xenograft growth in nude mice
[34]. Immunohistochemistry confirmed that the decrease in
tumour size was associated with KRAS degradation, with the
control groups exhibiting stronger KRAS expression than the
RC-U expressing group [34].
The UPP system has a high specificity and has the possibility
to eliminate oncogenic KRAS. With recent results arising about
the alternative pathway to the UPP system that involves ornithine
decarboxylase (ODC) and antizyme (AZ), the engineered targeted
KRAS oncogene degradation may be a highly efficient treatment
option for PDAC [34]. While these results are promising, the
mechanism of delivery and development of the RC-U engineered
protein into a therapeutic may be challenging.
Targeting mutated KRAS in pancreatic cancer
Targeting KRAS using RNA interference
KRAS mutations in PDAC
The activation of oncogenic KRAS appears to be one of the
preliminary events detected in pancreatic cancers. This discovery
makes KRAS a desired molecular target for the treatment of
pancreatic cancer [31]. However the direct targeting of KRAS was
not successful to date [32]. As an alternative approach, targeting
downstream-mediators of RAS-signalling are emerging with
most of them being in pre-clinical development stage. These
approaches are detailed below.
KRAS degradation by a programmed ubiquitin
ligase (E3)
Forced degradation of KRAS by the ubiquitin-proteasome pathway
O’ Reilly et al. 2015
Silencing of KRAS has proven to be effective in down-regulating
the proliferation of pancreatic cells. The development of a
controlled delivery system could enable the treatment of
pancreatic cancer using siRNA technology [35].
Technology developed by Khvalevsky and colleagues gave
rise to new hope with targeted drug delivery systems. A delivery
system known as Local Drug EluteR (LODER) was developed [36].
This ‘gradual-release’ polymer matrix was utilised for the treatment
of solid tumours in murine models. This siG12D LODER
was developed on the basis that most KRAS ‘gain-of-function’
mutations are of a replacement of the glycine (G) at position 12 to
aspartate (D) [36]. The siG12D LODER is a small, biodegradable
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Annals of Cancer Research
polymeric matrix which encapsulates the KRASG12D siRNA pancreatic cancer cell lines [38]. NF-κB represents a family of
drug. Its design features take into consideration the requirements transcription factors essential for regulation of the transcription
for slow, steady drug release and the resistance of the LODER of genes responsible for cellular functions such as apoptosis,
against rapid excretion [36].
inflammation and oncogenesis [39,40]. The NF-κB family consists
The functionality of the LODER was assessed in PANC-1 cells, of five members. These are as follows; cellular reticuloendotheliosis
containing KRASG12D and which also expressed luciferase [36]. viral oncogene homolog (c-Rel), RelB, RelA (p65), p59 (coded
Mice were injected with the PANC-1-Luc in the pancreas. The by NF-κB1 gene and its precursor is p105, which undergoes
mice had a laparotomy and two LODERs were inserted into the limited proteolysis to produce the functional, p50 fragment)
tumour masses. By measuring luciferase activity, it was found and p52 (coded by the NF-κB2 gene whose precursor is p100)
that the siG12D LODER reduced tumour growth in comparison [39]. These proteins all share a common amino-terminal Rel
to the controls [36].
homology domain (RHD), essential for dimerization, DNA
Immunohistochemical detection of KRAS confirmed that binding and inhibitor kappa B protein (IκB) interaction [39].
siG12D LODER reduced KRAS expression [36]. Utilising a LODER
cRel, RelA and RelB also possess a transcription activation
defeats many of the obstacles other approaches were facing. The domain (TAD) required for induction of gene expression. p50
tumour could be targeted where a stable, local release of the drug and p52 lack this domain and are only functional when associated
could be established [36]. This could be a potential mechanism to a TAD-containing family member [39].
for the specific targeting of pancreatic tumours.
There are two distinct pathways of NF-κB (Figure 1). The
canonical pathway leads to the activation of the p50/p65 dimer
Targeting scaffold-CRAF kinase interaction with and is typically activated in response to Tumour Necrosis Factor
RAS-ERK pathway
(TNF) [39]. When TNF binds to its receptors, it activates the
High activity of ERK1/2 detected in PDAC results from mutant IκB kinase (IKK) complex consisting of IKKα, IKKβ and NEMO.
KRAS [35]. Therefore, the targeting of RAS-downstream kinases, The complex phosphorylates inhibitor kappa B (IκB) leading to
such as RAF and MEK (activators of ERK), are currently being its ubiquitination and consequent degradation of and release of
tested as therapeutic targets [35]. Yet, obtaining an inhibitor, NF-κB from the inhibitory interaction with IκB [41]. The nonwhich is specific to the kinases, proves difficult due to the similar canonical pathway is activated by other TNF cytokines, such
catalytic domain structure that many kinases share [35]. Luan as CD40 and it is dependent on NF-κB inducing kinase (NIK)
and colleagues identified prohibitin (PHB), a scaffold protein, activation, as opposed to the IKK complex. NIK phosphorylates
which is required for the interaction between RAF and RAS [35]. p100, which initiates its processing to p52. This results in the
This interaction is essential for the activation of RAF and the accumulation of RelB/p52 and transcription of NF-κB target
downstream activation of the ERK pathway [35]. Another study by genes [41]. The main difference between the two NF-κB activation
Polier and colleagues identified that the binding of Rocaglamide pathways is that the canonical pathway triggers a quick, transient
(RocA), a naturally occurring compound, to PHB impairs NF-κB response, while the non-canonical pathway leads to a
the interaction between cRAF (RAF1) and PHB, and thus slower, maintained NF-κB signal [42].
blocking ERK1/2 activation in leukaemia cells [37].
Similarly to other tumours, the constitutive activation of NFIt was verified that PHB was abundantly present in pancreatic κB in pancreatic cancer is not primarily determined by mutations
cancer cells and silencing of PHB blocked EGF-induced phos- of genes involved in its regulation, but rather by pro-inflammatory
phorylation of ERK in PDAC cell lines suggesting that PHB is cytokines, such as TNF and IL-1α released by tumour-infiltrating
required for the activation of ERK1/2 in pancreatic cancer cells [35]. immune cells. Studies have demonstrated that IL-1α drives the
When the cells were treated with RocA, the phosphorylation activation of NF-κB in metastatic pancreatic cancer cell lines,
of cRAF and PHB in the membrane fraction was reduced [35]. which in turn induces expression and secretion of IL-1α from
The anti-tumour activity of RocA was further tested in severe the tumour cells, thus establishing a positive feedback loop for
immunodeficient mice with orthotopic AsPC-1 tumour cell constitutive NF-κB activation [43,44].
xenografts [35]. The mice were treated daily with RocA via
Interestingly, Bang and colleagues have shown that oncogenic
intraperitoneal injection. The mice treated with the RocA KRAS promotes constitutive NF-κB activation by inducing glycogen
exhibited a significant decrease in tumour growth compared synthase kinase 3α (GSK-3α). GSK-3α binds to the IKK-activating
to the vehicle-treated mice [35].
protein complex, consisting of transforming growth factor-beta
Targeting the PHB-CRAF interaction emerges as a new activated kinase 1 (TAK1) and TAK1-binding protein (TAB), and
potential mechanism for the inhibition of oncogenic KRAS, an stabilises it, thus enhancing canonical NF-κB activation. GSK-3α
alternative to the current unsuccessful direct inhibition of KRAS. can also enhance IKKα and NIK activity through which it also
RocA arises as a new potential approach, overcoming the lack of drives the non-canonical NF-κB pathway [45]. Upon constitutive
specificity than can be associated with generic kinase inhibitors. activation, NF-κB can inhibit pro-apoptotic signalling pathways.
Aberrant Nuclear Factor-κB pathway
Nuclear factor kappa B (NF-κB) is activated in over 70% of human
O’ Reilly et al. 2015
Targeting the NF-κB pathway in pancreatic cancer
The non-canonical pathway of NF-κB is often active in pancreatic
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Annals of Cancer Research
A
TNF
B
TNFR1
TAB2/3
KKβ Ub Ub Ub Ub
TAK1
NEMO
Gene expression
NF-κB
TRAF2 TRAF3
clAP
SHARPIN
clAP
Ub
Ub
Ub HOIP
Ub HOIL-1
IKKα
NIK
Ub
Ub
UbUb
TRAF2 TRAF3
Ub
Ub
Ub
Ub
UbUb
UbUb
clAP
TRAF2
Ub
Ub
Ub
Ub
CD40
R1P1
TRADD
CD40L
NIK
Proteosomal
degradation
Proteosomal
degradation
Gene expression
NF-κB
Figure 1. The canonical and the non-canonical NF-κB pathways.
(A) The canonical NF-κB pathway. Binding of TNF to TNFR1 triggers the recruitment of an adaptor
protein complex consisting of TRADD, TRAF2, cIAP1, cIAP2 and RIP1. cIAPs dimerise inducing
their E3 ligase activity. cIAPs add non-degradative (K11, K63) ubiquitin chains onto receptor
interacting protein kinase 1 (RIP1), themselves and other binding partners. RIP1 ubiquitination
initiates the binding of two kinase complexes, the IκB kinase complex and the TAK1-TAB2/3
complex, and another ubiquitnating protein complex, called LUBAC. The LUBAC complex contains
two regulatory subunits: SHANK-associated RH domain interactor (SHARPIN) and hemeoxidized IRP2 ubiquitin ligase 1 homolog (HOIL-1), and the catalytic subunit HOIL-1-interacting
protein (HOIP). LUBAC adds a linear ubiquitin chain to NEMO contributing to its activation.
Ubiquitination of RIP1 brings the IKK complex into proximity of the TAK1 kinase complex, leading
to phosphorylation and activation of IKKβ. IKKβ phosphorylation triggers IκBα phosphorylation
and subsequent proteasomal degradation and freeing the p65 and RelA NF-κB subunits, their
translocation to the nucleus and initiation of gene expression. (B)The non-canonical NF-κB pathway.
In resting conditions, cIAP1/2, TRAF2 and TRAF3 form a complex with NIK, the key regulator of
non-canonical NF-κB signalling. In the complex, NIK is brought to close proximity with cIAPs and
it becomes ubiquitinated with K48, degradative linkages that target it for proteasomal degradation.
Activation of TNF superfamily receptors such as CD40 leads to the recruitment of the cIAP/TRAF2/
TRAF3 complex to the receptor where the substrate specificity of IAPs change; instead of targeting
NIK, they ubiquitinate themselves, leading to their degradation and release of NIK allowing its
accumulation. NIK phosphorylates IKKα dimers, p100 is phosphorylated and its partial proteasomal
degradation yields the truncated and active p52 fragment. The p52 fragment dimerises with RelB
and translocates to the nucleus to drive NF-κB dependent gene expression.
cancers, and has been linked to mediating chemoresistance and
metastasis [39]. For example, NIK, the key IkB-phosphorylating
enzyme in the non-canonical NF-κB pathway has a high
expression levels in pancreatic cancer [39]. Its inhibition, in
theory, should prevent pancreatic cancer growth. To date, the
inhibition of NIK has not been overly successful. Problems with
the inhibitors proposed for NIK included a lack of specificity or
ineffectiveness in vivo [46]. A prime example of an unsuccessful
inhibitor of NIK is ‘pyrazolo[4,3-c]isoquinoline’. This inhibitor
was not specific to the inhibition of NIK. The canonical pathway
of NF-κB was also affected [47]. As a result, to date, there is
increasing focus on the targeting of binding partners associated
O’ Reilly et al. 2015
with NIK as detailed below.
Upregulation of TRAF2 expression
NIK is the key activator of the alternative NF-κB pathway [48].
NIK, when associated with IKKα, stimulates the non-canonical
NF-κB pathway by inducing the conversion of NF-κB2 (p100) to
p52 leading to the formation of the p52/RelB NF-κB heterodimer
[48]. TRAF3 (TNF receptor-associated factor 3) binds NIK
when there is no stimulus, recruiting NIK to the TRAF2/cIAP1/
cIAP2 complex [48]. Upon CD40 and other TNF receptor
engagement, essential for the mediation of several immune
and inflammation responses, the activated receptor recruits
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Annals of Cancer Research
the TRAF2/TRAF3/cIAP1/cIAP2 complex. When bound to the
receptor, cIAPs ubiquitinate TRAF3, instead of NIK, leading to
its degradation and release of NIK, now free to activate IKKα
and p100 processing (Figure 2) [48].
NIK expression and non-canonical NF-κB activity in
pancreatic cancer can be driven by GSK-3 induced by oncogenic
KRAS or, as a recent study highlighted may be the cause of
proteolytic degradation of the adaptor protein, TRAF2 [45].
TRAF2 downregulation is tightly associated with NIK
accumulation in high-grade adenocarcinomas. According to the
study of the Storz laboratory of 55 primary PDAC tissues, 83%
of the grade 3 adenocarcinomas showed low TRAF2 expression
with high NIK expression and high phosphorylated NIK levels
(on threonine 559 of NIK). None of the 10 healthy pancreatic
tissues showed this association [48].
Further studies showed that ectopic expression of TRAF2 in
PANC-1 PDAC cell line with normally very low TRAF2 expression
and constitutive NF-κB activity reduced NIK expression,
confirming the connection between TRAF2 deficiency and
overactivation of the non-canonical NF-κB pathway [48]. These
results highlight that by targeting this pathway, the proliferation
of pancreatic cancer cells could be decreased which may offer
a new approach for treatment. A potential therapeutic strategy
A
could be the inhibition of cIAP1 and cIAP2, the two ubiquitin
ligases associated with TRAF2, which ubiquitinate NIK in the
absence of a stimulus and degrade TRAF2 and/or TRAF3 upon
inflammatory cytokine stimulus [48].
It has to be noted that TRAF2 and IAPs have a dual role in
the canonical versus non-canonical NF-κB pathways, whereby
in the canonical pathway TRAF2 and IAPs participate in nondegradative ubiquitination reactions necessary for the assembly
of the NF-κB-activating complex, while they participate in NIK
degradation in the non-canonical pathway (Figure 1). Thus the
outcome of IAP inhibition on the balance and dynamics of NFκB activity is difficult to predict.
Inhibition of TAK1
Research conducted by Bang and colleagues has led to the
discovery of two downstream effectors as potential targets for
the inhibition of oncogenic KRAS. These are two kinases called
TGF-β–activated kinase 1 (TAK1) and glycogen synthase kinase
3 (GSK-3). Intriguingly, as described previous, these two kinases
also play an important role in the NF-κB pathway through which
they can enhance mutated KRAS-driven oncogenesis [45].
Transforming growth factor β activated kinase-1 (TAK1) is a
mitogen-activated kinase kinase kinase (MAP3K). When TAK1
B
Pancreatic cancer cell
Normal pancreatic cell
TRAF2 TRAF3
clAP
NIK
Ub
Ub
Ub
IKKα
TRAF2
Ub
Ub
Ub
Ub
NIK
p100
IKKα
P
p100
Ub
P
p52
TRAF2
degradation
TRAF2 stability
NIK proteasomal degradation
RelB
p52
PDAC cell
proliferation
Figure 2. Mechanism for maintenance of NIK in pancreatic cancer cells.
In normal pancreatic cells, the TRAF2/TRAF3/cIAP1/2 complex degrades NIK via
ubiquitination. In PDAC, TRAF2 degrades via ubiquitination, preventing the complex
formation, allowing for accumulation of NIK. NIK activates IKKα by phosphorylating it. Once
active, IKKα phosphorylates p100 that initiates its ubiquitination and limited proteolysis in
the proteasome generating the p52 fragment. p52 forms a heterodimer with RelB to drive the
expression of NF-κB target genes that promote cancer cell proliferation and disease progression.
(=:K48 linkage between ubiquitin units).
O’ Reilly et al. 2015
7
Annals of Cancer Research
binds to its partner proteins TAB1-4 (TGF-β activated kinase
binding protein 1-4) it undergoes autophosphorylation
and becomes active. Once active, TAK1 activates NF-κB by
phosphorylating and thus activating IKKβ.
While targeting of NF-κB itself is not feasible due to its
broad range of activities and its central role in the immune
system, inhibition of TAK1 may offer a safer strategy to inhibit
constitutive NF-κB activity in PDAC [45].
To establish the role of TAK1 in the activity in pancreatic
cancer, Bang and colleagues assessed the significance of the
TAK1-TAB1 complex and the effects of KRAS on the complex
in pancreatic cancer cells [45]. They showed that TAK1-TAB1
complex formation was low in normal human pancreatic ductal
epithelial cells (HPDE6) [45] and knockdown of KRAS in
KRAS-G12V-transformed HPDE6 cells (HPDEKR+) impaired
TAK1-TAB1 complex formation. In connection with a study
conducted by Melisi and colleagues [49], Bang and colleagues
highlighted the importance of TAK1 in NF-κB regulation in
cancer cells and that formation of the TAK1-TAB1 complex
was driven by oncogenic KRAS activity [45].
The effects of the first generation TAK1 inhibitor, 5Z-7
oxozeaenol, were analysed in pancreatic cancer cells expressing
mutant KRAS [45]. The results were consistent with the
hypothesis of Melisi [49]. Upon treatment with the TAK1
inhibitor, there was a significant decrease in the proliferation
of the MIA PaCa-2 pancreatic cancer cell line [45]. The 5Z-7oxozeaenol inhibitor triggered cell cycle arrest in the G2/M
phase of PANC-1 pancreatic cells indicating a role of TAK1 in
cell cycle progression [45]. Collectively, the data suggests that
the activation of TAK1 is driven by oncogenic KRAS and it is
involved in cell cycle progression [45]. Consequently, TAK1
emerges as a molecular target for the treatment of pancreatic
cancer. By inhibiting TAK1, cell-cycle arrest presumably results
in decreased cell proliferation [45,49]. The development of an
inhibitor, specific to the inhibition of TAK1 in pancreatic cancer,
could prove advantageous in the treatment of pancreatic cancer.
Inhibition of GSK-3
and thus emerges as a potential therapeutic target [45].
Other genetic alterations in pancreatic cancer
Epidermal growth factor receptor (EGFR) is encoded by the
c-ERBB-1 proto-oncogene. In a healthy pancreas, it is only
expressed in the islets of Langerhans. Overexpression of the
ERBB-1 gene is present in approximately 85% of pancreatic
cell lines in PDAC due to extensive gene transcription [31,50].
Pancreatic cancer progresses through a series of genetic
alterations. KRAS mutations and EGFR gene amplification are
the primary genetic alterations to occur, but these mutations
are usually followed by mutations in tumour suppressor genes,
such as p16. The inactivation of the TP53, SMAD4 (DPC4) and
BRCA2 genes are genetic alterations that occur in the late stages
of pancreatic cancer [31].
In pancreatic cancer, inactivation of tumour suppressor genes
(TSGs) has been shown to contribute to growth advantage
[51,52]. Loss of TSGs results from abnormalities such as allelic
deletion, mutations and chromosomal recombination [51,52].
The most frequent TSG mutation in PDAC is of p16, which is
inactivated in 95% of pancreatic cancers by mechanisms such
as homozygous deletion and loss of function mutation [53,54].
p16 (cyclin-dependent kinase inhibitor 2A/CDKN2A) plays an
important regulatory role at the G1-S phase checkpoint in the
cell cycle by binding to cyclin-dependent kinases[54]. TP53
(tumour protein p53) has many important cellular functions
including cell cycle control, stimulation of apoptosis and DNA
repair [51]. The loss of p16 and TP53 functionality encourages
cell survival and division of cells with damaged DNA, facilitating
the development of pancreatic cancer [51,54]. Another common
TSG mutation in PDAC is of DPC4 (SMAD4). Loss of DPC4
(deleted in pancreatic carcinoma, locus 4) typically occurs by
homozygous deletion and mutation. Mutations of DPC4 are
associated with poor prognosis and metastasis of pancreatic
cancer [54,55].
BRCA2 (breast cancer 2, early onset) is involved in homologous
recombination and thus its role is fundamental in the repair of
double-strand breaks during cell cycle division [56]. Biallelic
inactivation of BRCA2 occurs in approximately 7-10% of
pancreatic cancer. BRCA mutations are unlikely to be initiators
of pancreatic cancer, during the ontogenesis of PDAC other
genetic alterations occur before the inactivation of BRCA2 [56].
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase
with two isoforms: GSK-3α and GSK-3β. This kinase plays a key
role in cell cycle progression and apoptosis. GSK-3α promotes
constitutive IKK and NF-κB activity in pancreatic cancer by
targeting TAK1 [31]. To determine if GSK-3 is required to
preserve TAK1-TAB interactions, the effect of GSK-3 inhibition Targeting the other genetic alterations
was tested in MIA PaCa-2 cells [45]. It appeared that inhibiting As mention above, EGFR (HER1) is often overexpressed in
the activity of GSK-3α, but not GSK-3β, decreased TAK1 activity, pancreatic tumours. This overexpression of EGFR has been
preventing the formation of the TAK1-TAB1 complex [45]. In successfully targeted by the HER1/EGFR tyrosine-kinase inhibitor,
vivo mouse xenograft studies confirmed these results. Treatment erlotinib. Erlotinib used in combination with gemcitabine
of xenograft carrying mice with the GSK inhibitor AR-A014418 improved progression-free survival of patients with advanced
reduced tumour growth [45]. It appeared that upon inhibiting pancreatic cancer. The effects were modest but significant with the
GSK-3, the expression of proliferation promoting genes, such one year survival rate increasing from 17% with gemcitabine alone
as c-Myc and cIAP2, were reduced [45]. These studies showed to 23% with the combination of erlotinib and gemcitabine [57].
that GSK-3α plays a role in mediating the effect of oncogenic
BRCA2 also emerges as a potential molecular target for the
KRAS through the regulation and stabilisation of TAK1-TAB1 treatment of pancreatic cancer. BRCA2-mutated tumour cells,
O’ Reilly et al. 2015
8
Annals of Cancer Research
being impaired in homologous recombination DNA repair, rely
on alternative DNA repair pathways for survival. Inhibition of
these pathways, especially blocking the activity of the poly ADP
ribose polymerase (PARP) enzyme, induces synthetic lethality
in BRCA-mutant tumour cells. PARP inhibition results in the
transformation of single-stranded breaks to double-stranded
breaks, however in the absence of homologous recombination
repair, these lesions become cytotoxic, presenting an opportunity
for therapeutic intervention [58].
The synthetic lethal interaction between BRCA and PARP has
been further supported by the work of O’ Reilly and colleagues
who showed that the BRCA2 mutation is a biomarker predicting
the efficacy of genotoxic drugs for BRCA2 mutation-carrying
PDAC patients [59]. The phase Ib lead-in dose-finalisation
study showed a high activity of Veliparib, a PARP1 and PARP2
inhibitor, in combination with cisplatin and gemcitabine in
patients with BRCA2-mutated pancreatic adenocarcinoma. This
triple combination is now currently in phase II trial in BRCA/
PALB2-mutated patients with stage III/IV disease [60].
KRAS: Kirsten rat sarcoma viral oncogene homolog
MAPK: Mitogen-activated protein kinase
NF-κB: Nuclear Factor kappa B
TNF: Tumour Necrosis Factor
IκB: Inhibitor kappa B
IKK: Inhibitor kappa B kinase
NIK: NF-κB inducing kinase
IL-1α: Interleukin-1 alpha
TAK1: Transforming growth-factor beta activated kinase 1
TAB: TAK1-binding protein
EGFR: Epidermal growth factor receptor
TP53: Tumor protein p53
SMAD4: SMAD family member 4
BRCA2: Breast Cancer Type 2 Susceptibility Gene
TRAF2/3: TNF receptor-associated factor 2 and 3
cIAP1/2: Cellular inhibitor of apoptosis 1 and 2
GSK-3: Glycogen synthase kinase
UPP: Ubiquitin-proteasome pathway
RAF1 (cRAF): v-raf-1 murine leukaemia viral oncogene homolog 1
LODER: Local Drug EluteR
ERK: Extracellular signal-regulated kinase
MAPK: Mitogen-activated protein kinase
Conclusions
The authors declare that they have no competing interests.
Pancreatic cancer is resistant to traditional forms of cancer
treatments due to the high abundance of molecular alterations.
The development of therapies to target these mutations could
help increase patient response to the traditional treatments such
as chemotherapy and radiation. Targeting of the NF-κB pathway
and oncogenic KRAS only represents two of the many potential
targets for the treatment of pancreatic cancer. KRAS is so far
an undruggable protein and thus alternative approaches aim to
block the signalling either upstream or downstream of RAS. A
very promising upstream target was the farnesyl transferases
that create the lipid anchor of KRAS for membrane localisation.
In preclinical models, the farnesyl transferase inhibitors showed
great potency; however in clinical studies, their activity was far
less than anticipated. The approaches to target the downstream
segment of RAS signalling still hold promise. A significant
amount of data exists to support the evidence that both NF-κB
and KRAS are essential in transformation to pancreatic cancer
by proliferation and resistance to apoptosis. However, one
must consider that the targeting of the KRAS and NF-κB by the
processes described above are only in pre-clinical preformed on
mice models. The response of mice to the pre-clinical study to the
treatment may differ in human trials. Limited understanding of
the molecular pathogenesis of pancreatic cancer contributes to
previous failed attempts at targeting this disease. Extensive clinical
testing is required, but the discovery of these molecular targets
proposes new directions in the treatment of this pathogenic
disease. The use of these novel molecular targeted drugs in
combination with primary treatments, such as gemcitabine,
could prove beneficial in achieving a more effective treatment
for PDAC.
List of abbreviations
PDAC: Pancreatic Ductal Adenocarcinoma
O’ Reilly et al. 2015
Competing interests
Authors’ contributions
EO’R and ES collected the information, and prepared the manuscript
draft. CS and ES were responsible for conceptual framework and
revisions of the manuscript.
Acknowledgement
The research supporting this article has been funded by Science
Foundation Ireland (SFI) SIRG grant to ES (09/SIRG /B1575).
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