The Regulation of Constitutive NF-κB Activity by Glycogen Synthase Kinase-3 in Pancreatic Cancer
Willie Wilson III
A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics & Molecular Biology
Chapel Hill 2009
Approved by:
Dr. Al Baldwin
Dr. Gary Johnson
Dr. Leslie Parise
Dr. Adrienne Cox
Dr. Channing Der
ABSTRACT
WILLIE WILSON III: Regulation of Constitutive NF-κB Activity in Pancreatic Cancer by Glycogen Synthase Kinase-3 (Under the direction of Dr. Al Baldwin)
The development of pancreatic cancer chemotherapy has evolved into targeting the complex signaling pathways associated with disease progression. Recent focus has been made on targeting the constitutive activation of the transcription factor, NF-κB. However, advancement of this therapeutic strategy is dependent on fully understanding the elusive mechanisms underlying constitutive NF-κB activity in pancreatic cancer. Glycogen synthase kinase-3 has previously been implicated in regulating pro-survival NF-κB signaling in pancreatic cancer cells through an unknown mechanism. Moreover, TGF-β activated kinase
1 (TAK1) is a known regulator of NF-κB activity in human cancers, but its role in pancreatic cancer has yet to be established. In this report, we characterize GSK-3-dependent NF-κB regulation in pancreatic cancer cells and provide initial evidence that GSK-3 facilitates constitutive IκB kinase (IKK) activity. Our data also indicates that TAK1 is active in pancreatic cancer and contributes to constitutive NF-κB activation. Importantly, GSK-3 may be functionally linked to IKK through the phosphorylation of TAK1 at serine 412.
Collectively, we propose that constitutive NF-κB activity in pancreatic cancer cells is maintained by a novel GSK-3-TAK1-IKK signaling cascade. These data broadens our understanding of NF-κB regulation in pancreatic cancer and implicates GSK-3 and TAK1 as emerging therapeutic targets.
ii TABLE OF CONTENTS
LIST OF TABLES...... v
LIST OF FIGURES ...... vi
LIST OF ABBREVIATIONS...... viii
CHAPTER
I. INTRODUCTION
1.1 Summary...... 2
1.2 Pancreatic Ductal Adenocarcinoma...... 3
1.3 NF-κB Signal Transduction...... 5
1.4 Constitutive NF-κB Activation in Cancer ...... 8
1.5 TAK1-Dependent Regulation of NF-κB ...... 11
1.6 GSK-3-dependent Regulation of NF-κB ...... 14
1.7 GSK-3 Inhibitors as a Target Therapy for Cancer...... 18
1.8 Conclusions...... 20
References...... 31
II. MAINTENANCE OF CONSTITUTIVE IκB KINASE ACTIVITY BY GLYCOGEN SYNTHASE KINASE-3α/β IN PANCREATIC CANCER
2.1 Abstract...... 46
2.2 Introduction...... 47
2.3 Materials and Methods...... 49
iii 2.4 Results...... 53
2.5 Discussion...... 60
References...... 74
III. GLYCOGEN SYNTHASE KINASE-3 DRIVES CONSTITUTIVE TAK1- IKK SIGNALING IN PANCREATIC CANCER
3.1 Abstract...... 78
3.2 Introduction...... 79
3.3 Material and Methods ...... 82
3.4 Results...... 85
3.5 Discussion...... 91
References...... 102
IV. CONCLUSIONS & FUTURE DIRECTIONS
4.1 Conclusions & Future Directions...... 107
References...... 116
iv LIST OF TABLES
Table
1.1 Frequency of genetic mutations associated with pancreatic cancer...... 23
v LIST OF FIGURES
Figure
1.1 Accumulation of genetic mutations during pancreatic carcinogenesis...... 22
1.2 Domain organization of NF-κB family members...... 24
1.3 Canonical versus non-canonical NF-κB signal transduction...... 25
1.4 Domain organization of IKK and IκB family members ...... 26
1.5 Domain organization of TAK1 and TAB proteins ...... 27
1.6 TAK1-dependent signal transduction...... 28
1.7 Glycogen synthase kinase-3 isoforms...... 29
1.8 GSK-3 regulatory pathways and substrate targets...... 30
2.1 GSK-3 is required for constitutive NF-κB DNA binding...... 63
2.2 GSK-3 regulates NF-κB transcriptional activity ...... 64
2.3 Blockade of GSK-3 suppresses cell proliferation...... 65
2.4 IKK is required for constitutive NF-κB activation...... 66
2.5 Blockade of IKK suppresses cell proliferation ...... 67
2.6 GSK-3 inhibition suppresses constitutive IKK kinase activity...... 68
2.7 Model for GSK-3-dependent regulation of constitutive NF-κB activity in pancreatic cancer cells ...... 69
3.1 TAK1 is constitutively active in pancreatic cancer cells...... 95
3.2 TAK1 mediates pro-survival NF-κB activity in pancreatic cancer cells...... 96
3.3 Blockade of GSK-3 suppresses constitutive phosphorylation of TAK1 at serine 412...... 97
3.4 TAK1 phosphorylation at serine 412 is necessary to induce IKK phosphorylation...... 98
3.5 TAK1 phosphorylation at serine 412 is PKA-dependent ...... 99
vi
3.6 GSK-3 is not required for forskolin-induced TAK1 phosphorylation at serine 412...... 100
3.7 Model for inducible and constitutive TAK (S412) phosphorylation in pancreatic cancer cells...... 101
Supplemental Figures
2.1 GSK-3 regulates basal p65 DNA binding activity...... 70
2.2 Loss of GSK-3α attenuates cellular proliferation ...... 71
2.3 Loss of GSK-3α induces caspase-3/7 activity...... 72
2.4 Knock-down of IKK induces caspase-3/7 activity ...... 73
vii LIST OF ABBREVIATIONS
AKT v-akt murine thymoma viral oncogene homolog 1
AP-1 Activating Protein-1
APC Adenomatous polyposis coli
BRCA2 Breast cancer 2, early onset cAMP Cyclic adenosine 3'5' monophosphate
CDKN2A Cyclin-dependent kinase inhibitor 2A
COX-2 Cyclooxygenase-2
CREB cAMP response element binding protein
ELISA Enzyme linked immunosorbent assay
EMSA Electrophoretic mobility shift assay
GSK-3 Glycogen synthase kinase-3
GST Glutathione-S-transferase
HER2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2
HPDE Human pancreatic ductal epithelial cell
IκB Inhibitor of κB
IKK Inhibitor of κB kinase
IL-1β Interleukin 1 beta
JNK Jun N-terminal kinase
KRAS v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
LKB1 Liver kinase B1
MAP3K Mitogen activated protein kinase kinase kinase
MAPK Mitogen activated protein kinase
viii MKK Mitogen-activated protein kinase kinase
NBD NEMO-binding domain
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
PanIN Pancreatic intraepithelial neoplasia
PDA Pancreatic ductal adenocarcinoma
PGE2 Prostaglandin E2
PI3K Phosphoinositide-3-kinase
PKA Protein kinase A
RIP Receptor interacting protein
TAB TAK1-binding protein
TAK1 Transforming growth factor-β activated kinase-1
TCF/LEF T-cell-specific transcription factor/lymphoid enhancer factor
TGF-β Transforming growth factor-beta
TLR Toll-like receptor
TNF Tumor necrosis factor
TNFR TNF receptor
TP53 Tumor protein p53
TRAF Tumor necrosis factor receptor-associated factor
ix CHAPTER I
INTRODUCTION
1.1 Summary
The development of pancreatic cancer is characterized by the accumulation of various
oncogenic mutations which leads to the deregulation of key signaling pathways (Sarkar,
Banerjee et al. 2006). The constitutive activation of the transcription factor, NF-κB, is
among the many deregulated signaling pathways that have been associated with multiple
malignancies, including pancreatic cancer (Sarkar, Banerjee et al. 2006). Consequently,
there has been focus on targeting constitutive NF-κB activity in pancreatic cancer (Zhang
and Rigas 2006; Holcomb, Yip-Schneider et al. 2008). Advancement of this therapeutic
strategy is dependent on fully understanding the complex signaling events that drive NF-κB
activity. Here I provide new insight into the elusive signaling events that drive constitutive
NF-κB activity in pancreatic cancer and discuss glycogen synthase kinase-3 (GSK-3), TGF-β activated kinase-1 (TAK1), and IκB kinase (IKK) as potential therapeutic targets that can be utilized to disrupt this pathway. The following will provide a general overview of inducible
NF-κB signal transduction and evidence of regulation by GSK-3 and TAK1. Furthermore, I will review the functional significance of constitutive NF-κB activity during carcinogenesis and discuss the relevance to target this pathway in pancreatic cancer.
2 1.2 Pancreatic Ductal Adenocarcinoma
Pancreatic ductal adenocarcinoma (PDA) is the most common malignancy of the pancreas
and is the fourth leading cause of cancer related deaths in the United States (Jemal, Siegel et
al. 2008). Approximately 37,680 new cases of pancreatic cancer will be expected in 2008,
while 34,290 patients are estimated to die from this disease (Jemal, Siegel et al. 2008). Poor
early detection methods, aggressive metastatic potential, and chemotherapy resistance all
account for the dismal mortality rate associated with pancreatic cancer. The nucleoside
analog, gemcitabine, is among the most active single-agent chemotherapy currently used to
treat advanced stages of pancreatic cancer and is considered to be the first-line therapy for
patients (Burris, Moore et al. 1997; Abbruzzese 2002). Despite these efforts, the overall 5-
year survival rate for patients with pancreatic cancer still remains at 5% (Jemal, Siegel et al.
2008). Therefore, it is critical to advance our understanding of the molecular pathogenesis of pancreatic cancer in order to develop more targeted chemotherapies to treat this disease.
Extensive histological and genetic studies have characterized the pathogenesis of
pancreatic cancer into three early stages of pre-invasive lesions known as pancreatic
intraepithelial neoplasia (PanIN) (Fig. 1.1) (Hruban, Adsay et al. 2001; Kern, Hruban et al.
2001). The following PanINs are characterized by increasing degrees of architectural and
nuclear atypia: low-grade PanIN1A and PanIN1B, intermediate grade PanIN2, and high-
grade PanIN3 (Fig. 1.1). Various genetic alterations are known to accumulate throughout the
progression of PanIN lesions giving rise to invasive ductal adenocarcinoma (Fig 1.1). These
mutations include activation of proto-oncogenes (i.e. KRAS, AKT2, and HER2/ neu) and the
inactivation of tumor suppressors (i.e. p16/CDKN2A, TP53, DPC4/SMAD4, BRCA2, and
LKB1) (Table 1.1). KRAS, p16/CDKN2A, and TP53 are invariably among the most
3 frequently mutated genes associated with human pancreatic cancers (Table 1.1) (Hruban, van
Mansfeld et al. 1993; Redston, Caldas et al. 1994; Schutte, Hruban et al. 1997).
Recent advances in pancreatic cancer mouse models have revealed that these genetic events arise at distinct stages during the progression of PanIN lesions (Fig. 1.1). Gain-of- function point mutation in KRAS at codon 12 (G12V or G12D) occur early during tumor development and is considered to be the initiating genetic event in precursor PanIN1 lesions
(Moskaluk, Hruban et al. 1997). Moreover, endogenous expression of KRAS G12D in mouse pancreas progenitor cells induced early PanIN lesions which progressed into invasive ductal adenocarcinoma (Hingorani, Petricoin et al. 2003). Intermediate PanIN2 lesions are characterized by mutations, homozygous deletion, and promoter hypermethylation of the p16/CDKN2A locus (Moskaluk, Hruban et al. 1997; Schutte, Hruban et al. 1997; Wilentz,
Geradts et al. 1998). Furthermore, inactivation of the TP53 and DPC4/SMAD4 loci by loss- of-heterozygosity (LOH) or homozygous deletion are frequently observed later during tumor progression in PanIN3 lesions (Redston, Caldas et al. 1994; Rozenblum, Schutte et al. 1997).
Taken together, there has been significant advancement in our understanding of the genetic alterations that initiate and drive PanIN lesions into advanced ductal adenocarcinomas. As a result, new insight has emerged in comprehending the network of deregulated signal transduction pathways associated with pancreatic cancer. For example, constitutive signaling from PI3K/Akt, EGFR, COX-2, and Ras/Raf/MAPK pathways have all been implicated to play key roles in the development and progression of pancreatic cancers
(Sarkar, Banerjee et al. 2006). Interestingly, these deregulated pathways utilize molecular crosstalk with the transcription factor, NF-κB (Sarkar, Banerjee et al. 2006). Thus, focus has been on targeting constitutive NF-κB signaling as an alternative therapeutic strategy to treat
4 pancreatic cancer (Holcomb, Yip-Schneider et al. 2008). However, advancement of this strategy is dependent on fully understanding the signaling events that drive constitutive NF-
κB activation.
1.3 NF-κB Signal Transduction
NF-κB was originally discovered over 20 years ago as a nuclear factor that binds the immunoglobulin κ enhancer sequence in B-cells (Sen and Baltimore 1986). NF-κB has since been characterized as an inducible family of conserved dimeric transcription factors consisting of: RelA/p65, RelB, c-Rel, p52 (p100 precursor), and p50 (p105 precursor) (Fig.
1.2). NF-κB exists in cells as homo- or heterodimers and can function to either promote or repress gene transcription. The most studied NF-κB complex consists of the p65/p50 heterodimer (Ghosh, May et al. 1998). Figure 1.2 illustrates the domain organization of the
NF-κB family members. The N-terminal region of NF-κB consists of a highly conserved,
300 amino-acid, Rel homology domain (RHD) which promotes dimerization, nuclear localization, and DNA binding. A C-terminal transactivational domain (TAD) is located within RelA/p65, RelB, and c-Rel and facilitates NF-κB transcriptional activity upon inducible posttranslational modifications. Phosphorylation of RelA/ p65 at serines 536
(Mattioli, Sebald et al. 2004), 468 (Mattioli, Geng et al. 2006), and 276 (Zhong, Voll et al.
1998) has been reported to promote NF-κB transcriptional activity (Fig. 1.2). The C-terminal region of the precursor NF-κB members (p100 and p105) contains an inhibitor ankyrin repeat domain which functions to mask the nuclear localization sequence of NF-κB. This domain can undergo proteolytic cleavage, thus generating the mature p52 and p50 subunits.
5 Although processing of p105 to p50 is known to be constitutive, p100 to p52 processing is
tightly regulated (Xiao, Harhaj et al. 2001).
The inducible activity of NF-κB has been extensively studied since its discovery and has
been shown to play a central role in the regulation of diverse biological processes such as inflammation, immunity, development, cell proliferation, and survival (Ghosh and Karin
2002). The NF-κB target genes that drive these biological processes include cytokines (i.e.
IL-6, IL-12 TNF-α, and LTβ), chemokines (i.e. MCP1, IL-8, RANTES, and MIP-1α) , and adhesion molecules (i.e. ICAM and VCAM) (Ghosh and Karin 2002). The signaling events that regulate NF-κB-dependent gene expression can be classified into two pathways: canonical and non-canonical (Fig. 1.3).
In resting cells, NF-κB is rendered inactive and sequestered in the cytoplasm through
interaction with an inhibitor protein called IκB. IκB represents a family of conserved
proteins (IκBα, IκBβ, IκBε) which contain the aforementioned ankyrin repeat domain (Fig.
1.4B). Canonical NF-κB activity can be triggered by various stimuli including inflammatory cytokines, microbial infection, DNA damage, and mitogens (Pahl 1999). These stimuli
promote the activation of the IκB kinase (IKK) complex which consists of a regulatory
subunit (IKKγ) and two catalytic, serine/ threonine kinase subunits (IKKα and IKKβ). The domain organization of the IKK family members are illustrated in Figure 1.4A. Once this
complex is activated, IKKβ phosphorylates NF-κB-bound IκB proteins which leads to its
rapid ubiquitination and subsequent proteasomal degradation (Delhase, Hayakawa et al.
1999; Karin and Ben-Neriah 2000). Consequently, NF-κB can undergo nuclear translocation and bind to κB consensus sequences within target gene promoters.
6 Alternatively, non-canonical NF-κB signal transduction involves processing of the IκB- like p100 precursor (Fig. 1.3B). Prior to stimulation, RelB-containing NF-κB complexes are sequestered in the cytoplasm through interaction with p100. Diverse stimuli such as lymphotoxin (LTα, LTβ), B-cell activating factor (BAFF), CD40 ligand, tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), and RANKL trigger a signaling cascade which activates the NF-κB inducing kinase (NIK) (Xiao, Rabson et al. 2006). NIK drives the activation of IKKα homodimers and their association with p100 NF-κB complexes
(Bonizzi and Karin 2004). Notably, non-canonical NF-κB activation is independent of the canonical IKK complex (Senftleben, Cao et al. 2001) (Fig. 1.3B). IKKα subsequently phosphorylates p100, thereby leading to proteolytic processing and the generation of p52. As a result, activated RelB/p52 heterodimers can translocate to the nucleus to drive NF-κB- dependent gene transcription.
IKKα and IKKβ play pivotal roles in regulating the diverse pathways that lead to NF-κB activation. Despite structural and sequence similarities, IKKα and IKKβ have distinct, non- overlapping substrate specificities (Lee and Hung 2008). In addition to IκB, IKKβ also phosphorylates p65 at serine 536 which promotes nuclear translocation and enhanced NF-κB transactivation (Mattioli, Sebald et al. 2004). Moreover, NF-κB nuclear translocation can be mediated by IKKβ-dependent phosphorylation and subsequent proteolysis of p105
(Salmeron, Janzen et al. 2001). Contrary to IKKβ, IKKα possesses the unique ability translocate to the nucleus upon cytokine stimulation (Anest, Hanson et al. 2003). This function enables IKKα to target nuclear substrates required for NF-κB activation at target gene promoters. Nuclear IKKα has been shown to modulate chromatin structure at a subset
7 of NF-κB target genes by interacting with cyclic AMP-responsive binding protein (CBP), and phosphorylating histone H3 at serine 10 (Anest, Hanson et al. 2003; Yamamoto, Verma et al. 2003). Furthermore, IKKα also mediates the derepresssion of select NF-κB-responsive genes by promoting the phosphorylation and subsequent nuclear export of the co-repressor complex, SMRT (Hoberg, Yeung et al. 2004). Overall, IKKα and IKKβ can play distinct physiological roles in modulating various stages of NF-κB activity during an inflammatory response.
The complex signaling that modulates NF-κB activity must be tightly regulated in order to maintain normal physiological homeostasis. Notably, the deregulation of this process has linked NF-κB to chronic inflammatory diseases (i.e. rheumatoid arthritis and lupus) and tumorigenesis (Karin and Greten 2005; Okamoto 2006). There has been growing interest over the years in understanding the elusive mechanisms that drive NF-κB activity in pancreatic cancer and other human malignancies. The following section will review constitutive NF-κB signaling associated with cancer and therapeutic strategies currently being investigated to target NF-κB activity.
1.4 Constitutive NF-κB Signaling in Cancer
Chronic inflammation was first suspected to promote the development of cancer in the nineteenth century by one of the founding fathers of modern pathology, Rudolf Virchow
(Balkwill and Mantovani 2001). Decades later, NF-κB was implicated to be the missing link between chronic inflammation and cancer (Karin, Cao et al. 2002). Since then, constitutive
NF-κB activity has become well accepted as one of the many hallmarks of carcinogenesis.
8 Constitutive NF-κB activation can be observed in human tumor samples, as well in, various cancer cell lines. Breast cancer (Chua, Bhat-Nakshatri et al. 2007), colon cancer (Scartozzi,
Bearzi et al. 2007), lung cancer (Tew, Lorimer et al. 2008), melanoma (Yang, Pan et al.
2007), multiple myeloma (Annunziata, Davis et al. 2007), esophageal adenocarcinoma (Izzo,
Malhotra et al. 2007), and pancreatic cancer (Weichert, Boehm et al. 2007) are among the many human malignancies that have been demonstrated to have constitutive NF-κB activity.
Advancements in cancer mouse models have provided additional evidence connecting NF-
κB activity with the deregulated cellular processes associated with cancer (Naugler and Karin
2008). These processes include angiogenesis, metastasis, cell survival, cell cycle progression, and insensitivity to growth inhibition (Hanahan and Weinberg 2000).
The field of cancer research has made progress in understanding the molecular mechanism that drives the deregulation NF-κB activity. Evidence suggests IKK plays a central oncogenic role in regulating constitutive NF-κB activation in various human cancers
(Lee and Hung 2008). Constitutive IKK activity has been implicated in breast cancer
(Romieu-Mourez, Landesman-Bollag et al. 2001), prostate cancer (Gasparian, Yao et al.
2002), diffuse large B-cell lymphoma (DLBCL) (Lam, Davis et al. 2005), multi-drug resistant leukemias (Garcia, Alaniz et al. 2005) and pancreatic cancer (Liptay, Weber et al.
2003). The genetic ablation of IKK in cancer mouse models has also established a critical role for IKK during carcinogenesis. For instance, the deletion of IKKβ in enterocytes significantly reduces tumor formation in a mouse model of colitis-associated cancer (Greten,
Eckmann et al. 2004). Moreover, a Neu/ErbB2-driven breast cancer mouse model was shown to undergo significant growth suppression upon the genetic introduction of an inactive
9 IKKα mutant (Cao, Luo et al. 2007). Collectively, these reports and others underscore the
need to target IKK and NF-κB as a chemotherapeutic strategy.
Cancer researchers have improved their ability to identify the chemotherapeutic potential
of a broad range of synthetic and naturally-occurring chemical compounds (Steele, Sharma et
al. 1996). Notably, the chemopreventive effects of most of these agents have been linked to
the suppression of IKK and NF-κB through unclear mechanisms (Bharti and Aggarwal
2002). These chemopreventive agents include curcumin, retinoids, capsaicin, green tea
polyphenols, caffeic acid, phenethyl ester, and sulindac (Bharti and Aggarwal 2002).
Additionally, many pharmaceutical companies have made advances in developing small-
molecule inhibitors and cell-permeable peptides to target IKK activity. Due to the
predominant role IKKβ plays in canonical NF-κB signaling, one focus has been on developing selective IKKβ inhibitors. PS-1145 (Aventis Research and Technologies)
(Cilloni, Messa et al. 2006), BMS-345541 (Yang, Amiri et al. 2006), Compound A (Bayer
Yakuhin, Ltd.) (Duncan, Goetz et al. 2008), and BAY 11-7082 (Garcia, Alaniz et al. 2005)
are small-molecule inhibitors of IKKβ that suppress constitutive NF-κB activity and induce
cell death in various human cancer cell lines. The NEMO binding domain (NBD) peptide
was designed to interfere with IKK complex activation by blocking interaction between
IKKγ and the C-terminal region of IKKβ (May, D'Acquisto et al. 2000). NBD peptide was
recently reported to effectively suppress constitutive NF-κB activity and induce cytotoxicity
in human melanoma cell lines (Ianaro, Tersigni et al. 2009).
The discovery of new therapeutic strategies that target constitutive IKK/ NF-κB activity is
dependent on understanding the complex signaling events that drives this process. IKK and
subsequent NF-κB activity is initiated by a diverse set of upstream signaling events. The
10 versatility of these pathways enables molecular crosstalk between NF-κB and other signaling
pathways. The following sections will discuss the regulation of NF-κB by two versatile
serine threonine kinases, transforming growth factor β-activated kinase 1 (TAK1) and
glycogen synthase kinse-3 (GSK-3).
1.5 TAK1-dependent regulation of NF-κB
TAK1 belongs to the evolutionarily conserved mitogen-activated protein kinase kinase
kinase (MAP3K) family and was originally discovered to function within the transforming
growth factor-β (TGF-β) signaling pathway (Yamaguchi, Shirakabe et al. 1995). In addition to the TGF-β ligand family, this versatile serine/threonine kinase can be activated by a diverse set of stimuli such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α),
lipopolysaccharide (LPS), and latent membrane protein 1 (LMP1) (Irie, Muta et al. 2000;
Takaesu, Surabhi et al. 2003; Wan, Sun et al. 2004). Upon receptor activation, TAK1 can
initiate individual downstream signaling cascades culminating in the activation of IKK and
MAPKs (JNK and p38) (Karin, Liu et al. 1997; Takaesu, Surabhi et al. 2003). As a result,
NF-κB and AP-1 transcription factors are activated respectively, and promote expression of
various cellular stress and inflammatory-response genes.
TAK1 activity is dependent on various phosphorylation events and interaction with
essential signaling adapters called TAK1 binding proteins (TAB1, TAB2, and TAB3) (Fig.
1.5). TAB1 constitutively binds to the N-terminal activation loop of TAK1 and mediates
autophosphorylation at threonine residues (178 and 184) and serine residue 192 following
stimulation (Kishimoto, Matsumoto et al. 2000; Yu, Ge et al. 2008). TAB2 and TAB3 are zinc-finger domain-containing adapters that bind the C-terminus of TAK1 and promote
11 recruitment to activated receptors via K63-linked polyubiquitinated signaling adapters
(Kanayama, Seth et al. 2004; Besse, Lamothe et al. 2007). Notably, TAB proteins have been
demonstrated to play a crucial role in facilitating TNF-α and IL-1β-dependent activation of
NF-κB and MAPK pathways (Kishimoto, Matsumoto et al. 2000; Besse, Lamothe et al.
2007). In addition to TAB proteins, prostaglandin E2 was reported to synergistically enhance inducible TAK1 activity via protein kinase A (PKA)-dependent phosphorylation of
TAK1 at serine 412 (Kobayashi, Mizoguchi et al. 2005). Due to the versatility of TAK1 activation, other upstream signaling adapters are required to drive distinct downstream signaling events. The following will describe individual TAK1-mediated pathways triggered by TNF or IL-1/ Toll-like receptors (Fig. 1.6).
TNF-dependent activation of TAK1 is initiated by the rapid recruitment of TNF receptor
associated factors (TRAF2 and TRAF5) to TNF-α-bound receptors (Fig. 1.6A). TRAF2 and
TRAF5 function as E3 ubiquitin ligases and catalyze the development of polyubiquitin chains linked by lysine-63 (K63) of ubiquitin (Chen, Bhoj et al. 2006; Ea, Deng et al. 2006;
Adhikari, Xu et al. 2007). Unlike K48 polyubiquitinated proteins, K63 polyubiquitination does not prompt proteasomal degradation, but instead aids in scaffolding zinc finger domain- containing adapters (Adhikari, Xu et al. 2007). TRAF2/5 promotes the polyubiquitination of receptor interacting protein 1 (RIP1) (Ea, Deng et al. 2006). The TAK1-TAB complex is recruited to the K63 polyubiquitinated RIP1 via the aforementioned TAB2 and TAB3 subunits (Kanayama, Seth et al. 2004). Once activated, the TAK1 complex can elicit downstream activation of p38 MAPK via MKK3/6 and JNK via MKK4/7 (Raman, Chen et al. 2007). TAK1-mediated regulation of NF-κB is promoted by direct phosphorylation of
IKKβ (serines 177 and 181) which drives downstream canonical NF-κB activity (Takaesu,
12 Surabhi et al. 2003). Moreover, there is evidence that activation of the IKK complex by TNF is dependent on IKKγ (NEMO) recruitment to the K63 polyubiquitinated RIP1 (Ea, Deng et al. 2006).
Alternative to the pathway described above, TAK1 can also be activated by the sequential recruitment of MyD88, Tollip, and IL-1 receptor-associated kinase-1 (IRAK-1) signaling adaptors to the activated IL-1 or Toll-like receptors (Fig. 1.6B) (Wesche, Henzel et al. 1997;
Burns, Clatworthy et al. 2000; Yamamoto, Takeda et al. 2004). Subsequently, TRAF6 oligomers are recruited to the receptor complex. TRAF6 functions as an E3 ubiquitin ligase and catalyzes auto-K63-polyubiquitination (Lamothe, Besse et al. 2007). Similar to RIP1,
TRAF6 K63 polyubiquitination is critical for the recruitment and activation of the TAK1-
TAB1-TAB2/3 complex (Besse, Lamothe et al. 2007; Lamothe, Besse et al. 2007). IL-1R/
TLR-induced activation of the TAK1 complex promotes downstream NF-κB signaling through the phosphorylation of IKKβ, and p38-JNK MAPK signaling through the phosphorylation of MKK6 (Wang, Deng et al. 2001). Notably, K63 polyubiquitination plays a key role in IL-1R/ TLR and TNFR-dependent TAK1 signal transduction. Thus, NF-κB- regulated deubiquitinating enzymes (CYLD and A20) serve as critical negative feedback regulators of these pathways (Adhikari, Xu et al. 2007).
Deregulated TAK1 signal transduction has been associated with constitutive NF-κB activity in human cancer cell lines (Jackson-Bernitsas, Ichikawa et al. 2007; Bertrand,
Milutinovic et al. 2008; Xie, Wang et al. 2009). Evidence suggests that aberrant signaling from TNFR and Toll-like receptor 2 (TLR2) can facilitate constitutive TAK1 activation. An autocrine TNF-α feedback loop was reported to mediate constitutive NF-κB activity through the TRADD-TRAF2-RIP-TAK1-IKK pathway in human head and neck squamous cell
13 carcinoma (HNSCC) cell lines (Jackson-Bernitsas, Ichikawa et al. 2007). Treatment of
HNSCC cell lines with neutralizing TNF antibody significantly downregulated TAK1
signaling and suppressed cell proliferation (Jackson-Bernitsas, Ichikawa et al. 2007). TNF
autocrine feedback signaling can also drive constitutive RIP ubiquitination in the human
breast cancer cell line, MDA-MB-231 (Bertrand, Milutinovic et al. 2008). Additionally,
TLR2 overexpression was recently reported in MDA-MB-231 cells to promotes constitutive
TAK1/ NF-κB signaling and cell invasion (Xie, Wang et al. 2009). Taken together, TAK1
plays a key role in regulating the complex pathways leading to NF-κB activity in human
cancers and could be considered a novel therapeutic target.
1.6 GSK-3-dependent NF-κB Signal Transduction
Glycogen synthase kinase-3 (GSK-3) was discovered over 20 years ago as a serine/threonine kinase involved in the downregulation of glycogen biosynthesis through the
phosphorylation and inactivation of glycogen synthase (Embi, Rylatt et al. 1980).
Subsequently, GSK-3 was purified from rabbit skeletal muscle (Hemmings, Yellowlees et al.
1981), and molecular cloning revealed that this enzyme was encoded by two independent
genes, GSK-3α and GSK-3β (Woodgett 1990; Woodgett 1991) . GSK-3α and GSK-3β are
closely related mammalian isoforms that share 85% overall sequence identity and 93%
similarity within the catalytic domain (Ali, Hoeflich et al. 2001). A significant difference
between GSK-3 isoforms is observed in the N-terminal region where GSK-3α possesses an
extended glycine-rich tail (Fig. 1.7). Although purified GSK-3 isoforms exhibit similar
substrate properties (Woodgett 1991), evidence from GSK-3α and GSK-3β deficient mouse
models suggest non-overlapping substrate specificities (Hoeflich, Luo et al. 2000; MacAulay,
14 Doble et al. 2007). GSK-3α deficient mice exhibit enhanced glucose and insulin sensitivity
(MacAulay, Doble et al. 2007), while GSK-3β deficiency is embryonic lethal at day E13.5
due to liver degeneration (Hoeflich, Luo et al. 2000).
Unlike most protein kinases, GSK-3 is constitutively active in resting cells and is
primarily regulated through inhibitory pathways (Fig. 1.8). Evidence suggests that
constitutive GSK-3 activity is maintained by tyrosine auto-phosphorylation within the T-loop
kinase domain at residues Y216 in GSK-3β and Y279 in GSK-3α (Fig. 1.7) (Hughes,
Nikolakaki et al. 1993). Although T-loop tyrosine phosphorylation has been proposed to
mediate substrate phosphorylation, it is not strictly required for GSK-3 kinase activity
(Dajani, Fraser et al. 2001). In contrast, GSK-3 kinase activity can be inhibited through N- terminal serine phosphorylation at residues S9 and S21 in GSK-3β and GSK-3α respectively
(Fig. 1.7) (Cross, Alessi et al. 1995). This phosphorylation results in the folding of the N- terminus into the charged catalytic pocket of GSK-3. As a result, a pseudosubstrate is created that competitively blocks binding of true GSK-3 substrates (Doble and Woodgett
2003).
Various upstream signaling events have been shown to culminate to the phosphorylation
and inactivation of GSK-3 (Fig. 1.8). Insulin is among the most widely studied inducers of
GSK-3 serine phosphorylation. This pathway is driven by phosphoinositide 3-kinase-
dependent activation of Akt. Consequently, Akt promotes the serine phosphorylation and
inactivation of both GSK-3 isoforms (Cross, Alessi et al. 1995). Epidermal growth factor
(EGF) also promotes serine phosphorylation of GSK-3 through MAPK-dependent activation
of p90RSK (also termed MAPKAP-K1) (Saito, Vandenheede et al. 1994). Other GSK-3
inactivating kinases include p70 ribosomal S6 kinase (p70S6K) (Armstrong, Bonavaud et al.
15 2001), cAMP-activated protein kinase (PKA) (Li, Wang et al. 2000), and protein kinase C
(Fang, Yu et al. 2002). Alternative to S9/21 phosphorylation, GSK-3 can also be negatively regulated within the Wnt/ β-catenin pathway.
Canonical Wnt/ β-catenin signaling is an evolutionarily conserved pathway which plays a key role during development by regulating cell growth, differentiation, and polarity (Frame and Cohen 2001). GSK-3 is most recognized for its role in negatively regulating this pathway in resting cells by targeting β-catenin phosphorylation and subsequent proteasomal degradation (Aberle, Bauer et al. 1997). GSK-3 functions within a β-catenin “destruction complex” comprised of adenomatous polyposis (APC), axin/ conductin, and β-catenin
(Hinoi, Yamamoto et al. 2000). Following Wnt stimulation, the activated receptor (Frizzled) signals to Dishevelled (Dvl) which results in the inactivation of GSK-3 through an unknown mechanism (Frame and Cohen 2001). Consequently, nuclear accumulation of β-catenin mediates the transactivation of genes regulated by the T-cell factor (TCF)/ lymphoid enhancer factor (LEF) family of transcription factors (Novak and Dedhar 1999).
GSK-3 is a multi-tasking kinase that plays an intricate role in diverse signaling pathways.
As a consequence, GSK-3 is known to have an expanded range of substrate specificities (Fig.
1.8). The consensus motif for GSK-3 substrates is Ser/Thr-X-X-X-Ser/Thr (X represents any amino acid). GSK-3 has the unique ability to target substrates that have been previously phosphorylated (primed) at the C-terminal Ser/Thr residues of the consensus motif (Fiol,
Mahrenholz et al. 1987). For instance, the C-terminal priming serine residue of the GSK-3 substrate, glycogen synthase, is phosphorylated by casein kinase 2 (CK2) (Frame and Cohen
2001). Notably, the priming of GSK-3 substrates is not required, but has been shown to enhance the ability of GSK-3 to phosphorylate its substrates (Thomas, Frame et al. 1999). In
16 many cases, phosphorylation by GSK-3 results in the inactivation or proteasomal degradation of its substrate. These substrates include glycogen synthase, eIF2B translational elongation factor (Welsh and Proud 1993), β-catenin (Yost, Torres et al. 1996), Tau microtubule- associated protein (Hanger, Hughes et al. 1992), and cyclin D1 (Diehl, Cheng et al. 1998).
Moreover, GSK-3 can modulate gene expression by enhancing or suppressing the activity of various transcription factors: c-Jun (Boyle, Smeal et al. 1991), c-Myc (Pulverer, Fisher et al.
1994), nuclear factor of activated T-cells c (NFATc) (Beals, Sheridan et al. 1997), and cAMP response element binding protein (CREB) (Fiol, Williams et al. 1994). Interestingly, there has been an increasing body of literature implicating the role GSK-3 plays in regulating the activity of NF-κB.
The first evidence of GSK-3-dependent regulation of NF-κB was provided by James
Woodgett and colleagues (Hoeflich, Luo et al. 2000). This group genetically engineered a mouse model in which the ATP-binding loop of GSK-β was deleted, thus rendering an inactive kinase. Interestingly, embryonic lethality was observed in GSK-3β homozygous null mice between E13.5 and E14.5 days. Further characterization of this phenotype showed these animals succumbed to multifocal hemorrhagic degeneration in the liver due to TNF-α- dependent hepatocyte apoptosis. Notably, this phenotype is consistent with the homozygous deletion of the p65 subunit of NF-κB (Beg, Sha et al. 1995) and IKKβ (Li, Van Antwerp et al. 1999). To demonstrate GSK-3β-dependent NF-κB regulation, mouse embryonic fibroblasts from GSK-3β-deficient mice were also shown to be defective in TNF-α-induced
NF-κB activation (Hoeflich, Luo et al. 2000).
The exact mechanism of how GSK-3β regulates inducible NF-κB activity remains controversial. Reports have implicated GSK-3β to either regulate early stages of NF-κB
17 activation (IKK complex activation and IκB degradation) or later stages (NF-κB transcription activity at target gene promoters). Bharat Aggarwal and colleagues reported that GSK-3β null MEFs are defective in inducing IKK activity and IκBα phosphorylation/ degradation in response to TNF-α, IL-1β, and lipopolysaccharide (Takada, Fang et al. 2004).
In contrast with this report, work from our lab and others have suggested that GSK-3β deficiency, as well as pharmacological GSK-3 inhibition diminishes cytokine-induced NF-
κB activity at the transcriptional complex level (Hoeflich, Luo et al. 2000; Schwabe and
Brenner 2002; Steinbrecher, Wilson et al. 2005). In fact, we demonstrated that GSK-3β functions to regulate the transcription of a select sub-set of NF-κB target genes (MCP-1 and
IL-6) in response to TNF-α (Steinbrecher, Wilson et al. 2005). Further evidence showed that
GSK-3β can directly target the phosphorylation of p65 (amino acids 354-551), thus promoting NF-κB transactivation (Schwabe and Brenner 2002). Overall, GSK-3β plays an essential role in crossregulating cytokine-induced NF-κB activation through either an IKK- dependent or independent mechanism. Due to the known pro-survival functions of NF-κB, studies have also focused on the potential role that GSK-3 may play during carcinogenesis.
1.7 GSK-3 Inhibitors as a Targeted Therapy for Cancer
GSK-3 has been linked to deregulated signaling pathways leading to the pathogenesis of various human diseases. Consequently, GSK-3 has emerged over the years as a therapeutic target for Alzheimer’s disease, bipolar disorder, dementia, and noninsulin-dependent diabetes mellitus (NIDDM) (Eldar-Finkelman 2002). Moreover, there is increasing evidence linking aberrant GSK-3 signaling with the development of cancer. GSK-3 is generally considered a
18 tumor suppressor due to its role in suppressing the oncogenic effects of the Wnt/ β-catenin
pathway in colorectal cancer (Shakoori, Ougolkov et al. 2005). However, this paradigm has
been challenged by recent reports that suggest GSK-3 plays an oncogenic role by facilitating
cell growth, survival, and constitutive NF-κB activity. Advancement of these studies was
made possible by the development of pharmacological GSK-3 inhibitors.
Lithium was the first tool discovered and used to inhibit GSK-3 activity (Klein and
Melton 1996). However, the broad kinase specificity range of lithium has prompted
pharmaceutical companies to develop small molecule inhibitors with increased specificity for
GSK-3 (Cohen and Goedert 2004). SB 216763 (ATP-competitive, IC50 = 34.3 nM), SB
415286 (ATP-competitive, IC50 = 77.5 nM), AR-A014418 (ATP-competitive, IC50 = 104
nM), and TDZD-8 (non-ATP-competitive, IC50 = 2μM) are among the most recently
developed GSK-3 inhibitors that have been implicated to have therapeutic potential in human
cancers (Coghlan, Culbert et al. 2000; Martinez, Alonso et al. 2002; Bhat, Xue et al. 2003).
Notably, these inhibitors do not provide significant selectivity between GSK-3α and GSK-
3β isoforms due to sequence identity within the kinase domain (Coghlan, Culbert et al. 2000;
Martinez, Alonso et al. 2002; Bhat, Xue et al. 2003).
Studies within the past five years have implicated GSK-3 inhibitors as a potential targeted
therapy for prostate cancer, multiple myeloma, leukemia, and pancreatic cancer. Inhibition
of androgen-responsive, prostate cancer cell growth was observed following GSK-3
inhibition (SB 216763) and was associated with decreased androgen receptor function
(Rinnab, Schutz et al. 2008). Growth suppression and induction of apoptosis was also
reported in multiple myeloma cell lines following treatment with TDZD-8 (Zhou, Uddin et
al. 2008). Moreover, SB 216763 caused the destabilization of the cyclin-dependent kinase,
19 p27Kip, and induced cell cycle arrest in MLL leukemia cell lines (Wang, Smith et al. 2008).
Chronic lymphocytic leukemia cells were also sensitive to GSK-3 inhibition (AR-A014418)
due to epigenetic silencing of NF-κB-responsive survival genes (Bcl-2 and XIAP) (Ougolkov,
Bone et al. 2007). Further evidence of GSK-3-dependent constitutive NF-κB activity was
provided in pancreatic cancer cell lines (Ougolkov, Fernandez-Zapico et al. 2005; Ougolkov,
Fernandez-Zapico et al. 2006). These reports demonstrated that GSK-3 is active in pancreatic cancer cells, and GSK-3 inhibition (AR-A014418 and SB 216763) suppressed constitutive NF-κB reporter activation and target gene expression (Bcl-2, XIAP, Bcl-xL, and
Cyclin D1) (Ougolkov, Fernandez-Zapico et al. 2005). Furthermore, the suppression of constitutive NF-κB activity following GSK-3 inhibition correlated with decreased cell growth/ survival in both in vitro (Ougolkov, Fernandez-Zapico et al. 2005) and in vivo models (Ougolkov, Fernandez-Zapico et al. 2006). Taken together, these reports establish
GSK-3 inhibition as a potential therapeutic strategy for various human cancers. Although the
mechanism of action remains unclear, GSK-3 inhibitors may function partially by
suppressing constitutive, pro-survival signaling to NF-κB.
1.8 Conclusions
For more than a decade, Gemcitabine has been considered to be the standard cytotoxic chemotherapy to treat advanced stages of pancreatic cancer (Burris, Moore et al. 1997).
However, the clinical benefits of Gemcitabine are limited due to the chemoresistant nature of
this disease (Kim and Gallick 2008). The evolution of pancreatic cancer chemotherapy is
dependent on the development of agents that specifically target deregulated molecular
pathways associated with disease progression (Berlin and Rothenberg 2003). Pancreatic
cancer is among many human malignancies that utilize constitutive signaling to the
20 transcription factor, NF-κB. Therefore, chemotherapeutic agents that target constitutive IKK and subsequent NF-κB activity may be efficacious in the treatment of pancreatic cancer.
Advancement of this therapeutic strategy requires full understanding of the complex
molecular mechanisms that regulate constitutive IKK and NF-κB activity.
This chapter reviewed the critical roles GSK-3 and TAK1 play in promoting cytokine-
induced and constitutive NF-κB activity in human cancers. Importantly, GSK-3 (Ougolkov,
Fernandez-Zapico et al. 2005; Ougolkov, Fernandez-Zapico et al. 2006) and IKK (Li,
Aggarwal et al. 2004) have been reported to drive constitutive NF-κB activity and cell growth/ survival in pancreatic cancer. Moreover, deregulated TAK1 signaling has been
associated with various human cancers (Jackson-Bernitsas, Ichikawa et al. 2007; Bertrand,
Milutinovic et al. 2008; Xie, Wang et al. 2009) and may also be linked to constitutive IKK and NF-κB activity in pancreatic cancer. Despite the evidence of GSK-3-dependent NF-κB activation in pancreatic cancer, the mechanism of regulation remains elusive.
The remaining chapters will characterize the requirements of GSK-3 isoforms (GSK-3α
and GSK-3β), catalytic IKK subunits (IKKα and IKKβ), and TAK1 for maintaining
constitutive NF-κB activity and cell growth/ survival in pancreatic cancer cells.
Furthermore, I will investigate the potential link between these signaling components, and
propose that constitutive NF-κB activity is driven by a unique GSK-3-TAK1-IKK signaling
cascade. Collectively, these studies emphasize the significance of NF-κB signaling in
pancreatic cancer, and highlight the potential of GSK-3 and TAK1 as novel therapeutic
targets for this disease.
21
Figure 1.1. Accumulation of genetic mutations during pancreatic carcinogenesis. Pancreatic cancer progression is characterized by three stages of pancreatic intraepithelial neoplasia (PanIN-1A/B, PanIN-2, and PanIN-3) followed by invasive pancreatic ductal adenocarcinoma (PDA). The above “PanINgram” illustrates increasing degree of cytoplasmic and nuclei atypia used to classify each PanIN lesion (Maitra, Adsay et al. 2003). As indicated, genetic alterations in oncogenes (K-ras and Her2/ Neu) and tumor suppressors (p16, p53, DPC4, and BRCA2) arise during distinct stages during PanIN progression.
22
23
Figure 1.2. Domain organization of NF-κB family members. The evolutionarily conserved NF-κB family members indicated above exists as homo- or heterodimers. The N- terminal rel homology domain (RHD) mediates DNA binding and coupling with NF-κB family members. NF-κB transcriptional activity is facilitated by the C-terminal transactivational domain (TAD). The phosphorylation of p65 at the indicated sites has been shown to promote transcriptional activity. p100 and p105 exist as precursors consisting of an inhibitory ankyrin repeat domain which masks nuclear localization sequence within the RHD of p65, RelB, and c-Rel. Upon phosphorylation, p100 and p105 undergo proteolytic cleavage giving rise to mature p52 and p50 respectively.
24
Figure 1.3. Canonical versus non-canonical NF-κB signal transduction. (A) Canonical NF-κB signaling can be triggered by the indicated stimuli which converge on the activation of the IKK complex (IKKα, IKKβ, and IKKγ). Upon activation, IKK phosphorylates NF- κB-bound, IκBα at serines 32/ 36. Consequently, IκBα undergoes rapid ubiquitination and subsequent proteasomal degradation. Liberated NF-κB can undergo nuclear translocation and activate target gene expression. (B) Non-canonical NF-κB signaling can be initiated by the indicated stimuli which activate downstream NIK and subsequent IKKα homodimers. IKKα phosphorylates p100/ RelB heterodimers at serines 866/ 872. As a result, p100 undergoes proteolytic cleavage giving rise to an active p52/ RelB heterodimer.
25
Figure 1.4. Domain organization of IKK and IκB family members. (A) The IKK complex is composed of the indicated IKK family members: catalytic subunits (IKKα and IKKβ) and a regulatory subunit (IKKγ/ NEMO). IKKα and IKKβ consists of a kinase domain, leucine zipper (LZ) domain, helix-loop-helix (HLH) domain, and a NEMO-binding domain (NBD). LZ and HLH facilitates IKKα/β interaction and catalytic activity (Zandi, Rothwarf et al. 1997). Phosphorylation at serines 176/ 180 and 177/ 181 promotes catalytic activity in IKKα and IKKβ respectively. IKKα/β interaction with NEMO is dependent on the C-terminal NBD of IKKα/β and the coiled-coiled (CC) domain in NEMO (May, D'Acquisto et al. 2000). (B) Inhibitor of κB (IκB) protein family consists of IκBα, IκBβ, and IκBε. Ankyrin repeat domains mask nuclear localization sequence of NF-κB. PEST domain promotes proteasomal degradation upon phosphorylation at the indicated N-terminal serine residues.
26
Figure 1.5. Domain organization of TAK1 and TAB proteins. TAK1 consists of an N- terminal kinase domain which undergoes autophosphorylation at the indicated serine/ threonine residues upon activation. TAK1 is also phosphorylated at serine 412 in a PKA- dependent manner (Kobayashi, Mizoguchi et al. 2005). TAK1 exist in complex with TAK1- binding proteins (TAB1, TAB2, and TAB3). TAB1 interacts with N-terminal region of TAK1, while TAB2/3 interacts at the C-terminal region. TAB1 consists of an uncharacterized, N-terminal protein phosphatase 2C (PP2C) domain. TAB2/3 contains CUE and zinc-finger domains which facilitate interaction with polyubiquitin chains.
27
Figure 1.6. TAK1-dependent signal transduction. (A) TNFα-mediated TAK1 signaling is initiated by the recruitment of TRAF2/5 to activated TNF receptors. TRAF2/5 promotes recruitment and K63 polyubiquitination of RIP1. TAK1/ TAB complex is recruited to RIP1 polyubiquitin chains and subsequently activated by phosphorylation at serine 192 and threonines 178, 184, and 187. (B) IL-1β and LPS-mediated TAK1 signaling is initiated by recruitment of MyD88, Tollip, and IRAK1 signaling adapters to activated IL-1 or toll-like receptors. TRAF6 is subsequently recruited to the receptor complex and undergoes K63 autopolyubiquitination. TAK1/ TAB complex is recruited to K63 polyubiquitinated TRAF6. Activated TAK1 promotes downstream activation of NF-κB signaling through phosphorylation of IKKβ. TAK1 also drives JNK and p38 MAPK activation through the phosphorylation of MKK4/7 and MKK3/6 respectively.
28
Figure 1.7. Glycogen synthase kinase-3 isoforms. Glycogen synthase kinase-3 exist as two, closely related mammalian isoforms (GSK-3α and GSK-3β). Gly-Rich indicates the N- terminal glycine-rich region located in GSK-3α. GSK-3 activity is mediated by autophosphorylation within the kinase domain at tyrosine residues 279 and 216 in GSK-3α and GSK-3β respectively. GSK-3 activity undergoes inhibitory phosphorylation at N- terminal serine residues 21 and 9 in GSK-3α and GSK-3β respectively.
29
Figure 1.8. GSK-3 regulatory pathways and substrate targets. GSK-3 can be negatively regulated by a variety of upstream signaling events through the phosphorylation at serines 9 and 21 in GSK-3β and GSK-3α respectively. Wnts trigger phosphorylation-independent inhibition of GSK-3 through dishevelled. GSK-3 phosphorylates serine/ threonine residues in substrates that encompass the indicated substrate motif. Select substrates require a priming phosphorylation event by a secondary kinase to enhance GSK-3 substrate phosphorylation. The various substrates recognized by GSK-3 are indicated above.
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44 CHAPTER II
MAINTENANCE OF CONSTITUTIVE IκB KINASE ACTIVITY BY GLYCOGEN SYNTHASE KINASE-3 α/β IN PANCREATIC CANCER
This chapter has been adapted from: Willie Wilson III and Al Baldwin. Maintenance of constitutive IκB kinase activity by glycogen synthase kinase-3α/β in pancreatic cancer.
Cancer Research 2008. 68 (19): 8156-63. 2.1 Abstract
Constitutive NF-κB activation is among the many deregulated signaling pathways that are proposed to drive pancreatic cancer cell growth and survival. Recent reports suggest that glycogen synthase kinase-3β (GSK-3β) plays a key role in maintaining basal NF-κB target gene expression and cell survival in pancreatic cancer cell lines. However, the mechanism by which GSK-3β facilitates constitutive NF-κB signaling in pancreatic cancer remains unclear. In this report, we analyze the contributions of both GSK-3 isoforms (GSK-3α,
GSK-3β) in regulating NF-κB activation and cell proliferation in pancreatic cancer cell lines
(Panc-1 and MiaPaCa-2). We demonstrate that GSK-3 isoforms are differentially required to maintain basal NF-κB DNA binding activity, transcriptional activity, and cell proliferation in
Panc-1 and MiaPaCa-2 cells. Our data also indicate that IKK subunits are not equally required to regulate pancreatic cancer-associated NF-κB activity and cell growth.
Importantly, we provide the first evidence that GSK-3 maintains constitutive NF-κB signaling in pancreatic cancer by regulating IKK activity. These data provide new insight into GSK-3-dependent NF-κB regulation, and further establishes GSK-3 and IKK as potential therapeutic targets for pancreatic cancer.
46 2.2 Introduction
Pancreatic cancer represents the fourth leading cause of cancer-related death in the United
States with a five-year patient survival rate of 5% (Jemal, Siegel et al. 2007). Moreover, an
estimated 33,370 patients were expected to die from this disease in 2007 (Jemal, Siegel et al.
2007). The dismal mortality rate from pancreatic cancer stems from its aggressive metastatic
nature and its ability to resist conventional chemotherapies (Sarkar, Banerjee et al. 2006).
Therefore, expanding our knowledge of the complex molecular pathways responsible for the development of pancreatic cancer is critical for the discovery of new therapeutic strategies.
Deregulated NF-κΒ and glycogen synthase kinase-3 signaling are among the many pathways that have been implicated in the pathogenesis of pancreatic cancer.
NF-κΒ represents a family of evolutionarily conserved transcription factors consisting of
five members: c-Rel, RelA (p65), RelB, p50 (NF-κB1/p105 precursor), and p52 (NF-
κB2/p100 precursor) (Ghosh, May et al. 1998). The most studied NF-κΒ complex consists
of the p65/p50 heterodimer. In resting cells, NF-κΒ is rendered inactive within the
cytoplasm through association with inhibitory IκΒ proteins. Various inflammatory stimuli
can trigger the activation of the IκΒ kinase (IKK) complex which consists of a regulatory
subunit (IKKγ) and two catalytic subunits (IKKα and IKKβ) (Zandi, Rothwarf et al. 1997).
Upon IKK activation, IκΒ is phosphorylated and subsequently targeted for rapid proteosomal degradation thus liberating NF-κΒ for nuclear translocation, enhanced DNA binding and transcriptional regulation (Karin and Ben-Neriah 2000).
Constitutive activation of NF-κΒ has been characterized in numerous human cancers and
is associated with regulating genes that control cell survival, proliferation, metastasis, and angiogenesis (Aggarwal 2004). Importantly, constitutive NF-κΒ activation has been
47 observed in 70% of human pancreatic cancers as well as in human pancreatic cancer cell lines and animal models (Liptay, Weber et al. 2003; Li, Aggarwal et al. 2004; Zhang and
Rigas 2006). Studies have also demonstrated constitutive IKK activity to play a key role in regulating cell survival and cell cycle progression in multiple in vitro pancreatic cancer models (Liptay, Weber et al. 2003; Schneider, Saur et al. 2006). Thus, there has been growing interest in utilizing IKK as a chemotherapeutic target for pancreatic cancer.
Glycogen synthase kinase-3 (GSK-3) is a serine/theronine kinase that exists as two highly similar mammalian isoforms (GSK-3α and GSK-3β) (Woodgett 1990; Woodgett 1991).
GSK-3 is recognized for its role in downregulating β-catenin, thus suppressing the transcriptional activity of T-cell-specific transcription factor (TCF)/lymphoid enhancer factor
(LEF) complexes within the Wnt/β-catenin pathway (Ali, Hoeflich et al. 2001). Numerous reports have subsequently demonstrated involvement of this multifunctional kinase in regulating a variety of transcription factors involved in cancer progression, including NF-κB
(Fiol, Mahrenholz et al. 1987; Boyle, Smeal et al. 1991; Pulverer, Fisher et al. 1994; Beals,
Sheridan et al. 1997; Ross, Erickson et al. 1999; Ali, Hoeflich et al. 2001). A GSK-3β deficient mouse model provided the first evidence of GSK-3-dependent NF-κB regulation
(Hoeflich, Luo et al. 2000). These data show that the loss of GSK-3β results in defective
NF-κB signaling in response to TNF-α. Furthermore, we previously reported that GSK-3β specifies promoter-specific recruitment of p65/RelA to NF-κB-dependent genes in response to TNF-α (Steinbrecher, Wilson et al. 2005). A previous report has also implicated GSK-3β in playing a critical role in regulating constitutive NF-κB reporter activity and target gene expression within in vitro pancreatic cancer models (Ougolkov, Fernandez-Zapico et al.
48 2005). However, the mechanism by which GSK-3β drives constitutive or inducible NF-κB has not been characterized.
Despite the structural similarity between GSK-3α and GSK-3β, evidence suggests that these isoforms are not functionally redundant in regulating NF-κB (Hoeflich, Luo et al. 2000;
Liang and Chuang 2006). In this report, we characterize the individual roles that each
GSK-3 isoform play in maintaining constitutive NF-κB activity and cell proliferation in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2). We show that both GSK-3 isoforms can function to regulate basal NF-κB DNA binding and transcriptional activity, whereas
GSK-3α predominantly controls cell growth and survival. Our data also demonstrate that
IKKα and IKKβ exhibit different requirements to drive constitutive NF-κB activity in a pancreatic cancer cell type-dependent manner. Additionally, we provide the first evidence that links GSK-3 to constitutive IKK activity in pancreatic cancer cells.
2.3 Materials and Methods
Cell Culture and Reagents. Panc-1 (CRL-1496) and MiaPaCa-2 (CRL-1420) pancreatic cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA).
Panc-1 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 100 units/ml penicillin/ streptomycin.
MiaPaCa-2 cells were maintained in DMEM supplemented with 10% fetal bovine serum and
2.5% horse serum. Cells were cultured in DMEM supplemented with 0.5% fetal bovine serum for 24 hour prior to experimentation. All cell culture reagents were obtained from
Invitrogen (Carlsbad, CA). The following antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA): p65 (SC-109), p50 (SC-7178), GSK-3α/β (SC-7291), β-
49 tubulin (SC-9104), and GST (SC-33613). IKKα clone 14A231 and IKKβ clone10AG2
antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): phospho-p65
(serine 536), phospho-glycogen synthase (serine 641), glycogen synthase, cleaved caspase-3
(Asp 175), and caspase-3. TNF-α was purchased from Promega (Madison, WI). GSK-3
inhibitors (AR-A014418 and SB216763) were obtained from Sigma-Aldrich (St. Louis,
MO). The IKKβ inhibitor (Compound A) was provided by Bayer Healthcare (Wuppertal,
Germany).
Small RNA interference. The following siRNA (siGenome SMARTpool) was obtained
from Dharmacon (Layfayette, CO) as a pool of four annealed double-stranded RNA
oligonucleotides: IKKα (M-003473-02), IKKβ (M-003503-03), GSK-3α (M-003009-01),
GSK-3β (M-003010-03), and non-targeting control #3 (D001201-03). In brief, cells were
cultured to 70% confluency in six-well plates. Dharmafect 1 transfection reagent
(Layfayette, CO) was used to transfect 100nM siRNA according to manufacturer’s
instruction.
Electrophoretic mobility shift assay (EMSA) and NF-κB DNA binding ELISA.
EMSA and NF-κB super-shift analysis was performed on nuclear extracts as previously described (Steinbrecher, Wilson et al. 2005) using 32P-labeled oligonucleotide probe corresponding to an NF-κB site within the MHC class I promoter region. Relative p65 DNA binding activity was quantified using the TransAM NF-κB p65 transcription factor assay kit
(Active Motif, Carlsbad, CA) according to manufacturer’s instructions. DNA binding activity was measured in triplicate at 450nm wavelength on a Versamax Microplate Reader
(Molecular Devices Corp., Sunnyvale, CA)
50 Western blot analysis. Whole-cell lysates were prepared on ice using Mammalian
Protein Extraction Reagent (M-PER) (Pierce Biotechnology, Rockford, IL) according to
manufacturer’s instructions. Cytoplasmic extracts were prepared as previously described
(Mayo, Wang et al. 1997). Protein extracts were quantified by Bradford assay (Bio-Rad
Laboratories, Hercules, CA) and analyzed by SDS-PAGE as previously described
(Steinbrecher, Wilson et al. 2005).
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium (MTS tetrazolium) cellular proliferation assay. Cells were seeded in
triplicate at 3 X 103 cells per well (96-well plate) and cultured in the presence or absence of
GSK-3 or IKKβ inhibitors at the indicated time course. Alternatively, cells were transiently
transfected with appropriate siRNA and cultured at the indicated time points post-
transfection. At the end of each time point, MTS tetrazolium compound (Promega, Madison,
WI) was added and absorbance was read at 490nm on a Versamax Microplate Reader
(Molecular Devices Corp., Sunnyvale, CA)
Dual-luciferase reporter assay. Cells were seeded in triplicate at 2 X 104 cells per well
(24-well plate), transfected with the appropriate siRNA as described above, and cultured for
24 hours. After siRNA transfection, cells were co-transfected with 200ng of luciferase
reporter construct containing tandem NF-κB binding sites from the MHC class I promoter
region and 5ng pRL-TK Renilla luciferase construct (Promega, Madison, WI) using Fugene6 transfection reagent (Roche Applied Science, Indianapolis, IN). Cells were cultured for an
additional 24 hours, harvested in passive lysis buffer, and analyzed according to Dual-
Luciferase Assay System protocol (Promega, Madison, WI). Relative light units were
51 measured on an Lmax Microplate Luminometer (Molecular Devices Corp., Sunnyvale, CA)
and normalized to pRL-TK Renilla luciferase light units.
Real-time PCR. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia,
CA) according to manufacturer’s instructions. Two micrograms of RNA was reverse transcribed into cDNA using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad,
CA). Real-time PCR was performed and analyzed as previously described (Steinbrecher,
Wilson et al. 2005) using TaqMan Gene Expression Assay primer-probe sets (Applied
Biosystems, Foster City, CA) for IκBα (Hs00153283_m1) and Bcl-xL (Hs00236329_m1).
IKK kinase assay. Whole-cell lysate were prepared on ice for 45 minutes in the
following lysis buffer containing 20mM Tris (pH 8.0), 500mM NaCl, 0.25% Triton-X100,
1mM EDTA, 1mM EGTA, 1mM dithiothreitol (DTT), 1X protease inhibitor (Roche Applied
Science, Indianapolis, IN), and 1X phosphatase inhibitor cocktail 1 (Sigma-Aldrich, St.
Louis, MO). IKK complexes were immunoprecipitated from 500μg protein extract using an
IKKα antibody (Upstate Biotechnology). An in-vitro kinase assay was performed and analyzed as previously described (Steinbrecher, Wilson et al. 2005). GST-IκBα substrate
phosphorylation was visualized by autoradiography and quantitated using Image Quant
Version 5.2 software (Molecular Dynamics, Sunnyvale, CA).
Luminescent-based caspase-3/7 activity assay. Cells were plated in triplicate at 2 x 103
cells per well in white-walled 96-well plates (Becton Dickinson, Franklin Lakes, NJ). Cells
were transiently transfected with siRNA as described above. Caspase-3/7 activity was
measured at 48, 72, and 96 hours post-transfection using the Caspase-Glo 3/7 assay
(Promega, Madison, WI) according to manufacturer’s instructions. Caspase-Glo 3/7 assay
utilizes a caspase-3/7 tetrapeptide DEVD substrate which produces a luminescent signal
52 upon cleavage. Relative light units were measured on an Lmax Microplate Luminometer
(Molecular Devices Corp., Sunnyvale, CA).
Statistical analysis. Prism software (GraphPad Software, Inc. San Diego, CA) was used
for statistical analysis of data. An unpaired t test was used to evaluate differences between
group means. P values less than 0.05 were considered to be statistically significant.
2.4 Results
GSK-3 isoforms regulate constitutive NF-κB activity in pancreatic cancer cells. A
recent study emphasized the role GSK-3β plays in maintaining constitutive NF-κB reporter
and target gene expression in pancreatic cancer cells (Ougolkov, Fernandez-Zapico et al.
2005). However, this report did not address the individual requirements of both GSK-3 isoforms in regulating NF-κB. Furthermore, the mechanism by which GSK-3 regulates NF-
κB activity requires understanding. Here we have analyzed constitutive NF-κB DNA binding and transcriptional activity in two well-known in vitro pancreatic carcinoma models
(Panc-1 and MiaPaCa-2). As shown in Fig. 2.1A, constitutive NF-κB DNA binding activity is detected in Panc-1 cells through EMSA analysis of nuclear extracts. This form of NF-κB activity is shown to be the p65/p50 heterodimer through supershift analysis (Fig. 2.1A).
EMSA analysis of MiaPaca-2 cells demonstrated constitutive DNA binding of p65/p50 heterodimers as well as p50 homodimers (Fig. 2.1A). Cells were treated with two structurally distinct pharmacological inhibitors of both GSK-3 isoforms (AR-A014418 and
SB-216763) to determine a potential requirement of GSK-3 in controlling constitutive NF-
κB DNA binding activity. Previously reported treatment duration and inhibitor concentrations (Ougolkov, Fernandez-Zapico et al. 2005) were used in our studies.
53 Treatment with either GSK-3 inhibitor for 24 hours at 15 and 25μM reduces p65/50 DNA
binding in both cell lines (Fig. 2.1B). DNA binding activity of p50 homodimers was largely
unaffected by GSK-3 inhibition in MiaPaCa-2 cells. To ask whether direct inhibition of
GSK-3 isoforms would affect NF-κB constitutive DNA binding activity in pancreatic cancer
cells, RNA interference of GSK-3α and GSK-3β was utilized. Western blot analysis
confirms that GSK-3 RNA interference was isoform-specific and significantly reduced GSK-
3 protein levels relative to non-targeting siRNA control (Fig. 2.1C). Loss of either GSK-3
isoform reduced constitutive NF-κB DNA binding in both cell lines (Fig. 2.1D). Moreover,
an ELISA-based DNA binding assay demonstrated a significant reduction of p65 binding
activity following the knock-down of either GSK-3 isoform (Supplemental Fig. 2.1)
To determine whether GSK-3 isoforms play roles in regulating NF-κB transcriptional
activity, we measured the effect of GSK-3 RNA interference on basal NF-κB-luciferase
reporter activity. Our data show that knock-down of either GSK-3 isoform resulted in a significant reduction of constitutive NF-κB-luciferase reporter activity in Panc-1 and
MiaPaCa-2 cells (Fig. 2.2A). Next, we examined whether GSK-3-dependent regulation of
reporter activity corresponded with the expression of two NF-κB-regulated genes (IκBα and
Bcl-xL). Loss of GSK-3β alone and both GSK-3α/β significantly suppressed expression of
Bcl-xL (Fig. 2.2B). Furthermore, knock-down of either GSK-3 isoform alone or combined
GSK-3α/β resulted in reduced expression of IκBα (Fig. 2.2B). Serine 536 phosphorylation
of the NF-κB subunit, p65/RelA, correlates with its transcriptional activity (Mattioli, Sebald
et al. 2004). To determine whether the GSK-3 isoforms regulate p65 phosphorylation, RNA
interference was used. Extracts were prepared from cells treated with control siRNA or
siRNA for either GSK-3 isoform. Consistent with the elevated NF-κB activity in Panc-1 in
54 MiaPaCa-2 cells, we also observe constitutive serine 536 phosphorylation of p65 (Fig. 2.2C).
RNA interference of GSK-3α or combined GSK-3α/β reduced p65 phosphorylation in Panc-
1 cells (Fig. 2.2C). Conversely, knock-down of GSK-3β had a greater effect on basal p65
phosphorylation in MiaPaCa-2 cells (Fig. 2.2C). Taken together, these data suggest that both
GSK-3 isoforms play individual roles in maintaining constitutive NF-κB DNA binding and
transcriptional activity in Panc-1 and MiaPaCa-2 cells.
GSK-3α promotes pancreatic cancer cell growth and survival. Constitutive NF-κB
activity has been reported to play mitogenic and anti-apoptotic roles in pancreatic cancer cell
lines (Liptay, Weber et al. 2003; Li, Aggarwal et al. 2004). Pharmacological GSK-3
inhibition and GSK-3 RNA interference were used to determine a role for GSK-3 in
controlling proliferation and survival of Panc-1 and MiaPaCa-2 cells. Consistent with a previous report (Ougolkov, Fernandez-Zapico et al. 2005), GSK-3 inhibition (AR-A014418) decreased growth of Panc-1 cells in a dose-dependent manner (5, 15, 30μM) over a 96 hour
time course (Fig. 2.3A). Similar results were observed in MiaPaCa-2 cells (data not shown).
Next, we used RNA interference to determine the individual requirements for GSK-3α and
GSK-3β on cell viability. We observed a significant reduction in Panc-1 cell growth following GSK-3α RNA interference at 96 hours post siRNA transfection when compared to non-targeting control and GSK-3β siRNA (Fig. 2.3B). Similar results were observed in
MiaPaCa-2 cells (data not shown). Moreover, the knock-down of GSK-3α in Panc-1 cells resulted in a significant reduction in the total number of cells at 72 and 96 hours post siRNA transfection (Supplemental Fig. 2.2A). Western blot analysis confirmed knock-down of
GSK-3 isoforms at each time-point post siRNA transfection (Supplemental Fig. 2.2B).
Importantly, we did not observe complete GSK-3 knock-down until 72 and 96 hours post
55 siRNA transfection (Supplemental Fig. 2.2B). These data could explain why the effects of
GSK-3α knock-down were only seen at later time-points post siRNA transfection.
The induction of caspase-3 cleavage was also measured to determine whether the loss of
cell viability following the disruption of GSK-3 was due to apoptosis. An increase in
caspase-3 cleavage following 30μM pharmacologic GSK-3 inhibition was observed over 24
and 48 hours in Panc-1 (Fig. 2.3C). The efficiency of GSK-3 inhibition was indicated by the
reduced basal phosphorylation of a GSK-3 substrate, glycogen synthase at serine 461 (Fig.
2.3C). Moreover, GSK-3α knock-down induced caspase-3 cleavage 48 hours post-
transfection of siRNA (Fig. 2.3D). Similar results were observed in MiaPaCa-2 cells (data
not shown). A luminescent caspase-3/7 assay was used to confirm a significant increase in
caspase activity following the knock-down of GSK-3α or combined GSK-3α/β in Panc-1
cells (Supplemental Fig. 2.3). Notably, pharmacologic GSK-3 inhibition was more effective
than GSK-3 knock-down in suppressing cell growth and inducing caspase-3 cleavage. These
observations could indicate unknown off-target effects of pharmacologic GSK-3 inhibition
(Fig. 2.3, see discussion). Taken together, we show that the loss of GSK-3α is more efficient
than GSK-3β in suppressing cell growth and survival in Panc-1 and MiaPaCa-2 cells.
Importantly, these observations correlate with the ability of GSK-3α to regulate constitutive
NF-κB activity.
Constitutive NF-κB activity in pancreatic cancer cells is IKK-dependent. In addition
to the studies shown above relative to GSK-3, studies have also shown IKK to be essential in regulating constitutive NF-κB signaling in pancreatic cancer cells (Liptay, Weber et al. 2003;
Li, Aggarwal et al. 2004; Schneider, Saur et al. 2006). To confirm these findings, NF-κB
DNA binding activity was analyzed in the presence of an established small molecule IKKβ
56 inhibitor (Compound A) (Ziegelbauer, Gantner et al. 2005). Compound A treatment for 24
hours showed dose-dependent suppression of constitutive NF-κB DNA binding activity in
Panc-1 and MiaPaCa-2 cells (Fig. 2.4A). We note that IKKβ inhibition is more effective in
blocking constitutive NF-κB DNA binding activity in MiaPaCa-2 cells. Next, RNA
interference to IKKα and IKKβ was utilized to determine the individual requirements of IKK
subunits for constitutive NF-κB DNA binding activity and transcriptional activation. The
knock-down of IKK subunits was specific and significantly reduced protein levels relative to
non-targeting siRNA control (Figure 2.4B). We observed a decrease in p65/p50 DNA binding activity following the knock-down of either IKK subunit in MiaPaCa-2 cells (Fig.
2.4C). Interestingly, knock-down of IKKα alone or combined IKKα/β knock-down caused
significant loss of NF-κB DNA binding activity in Panc-1 cells (Fig. 2.4C). The individual
role that each IKK subunit plays in regulating basal NF-κB transcriptional activity was
measured by an NF-κB reporter assay. RNA interference against either IKK subunit caused
substantial suppression of reporter activity in both Panc-1 and MiaPaCa-2 cells (Fig. 2.4D).
However, the suppressive effect of IKKα and combined IKKα/β knock-down were stronger
than IKKβ knock-down alone (Fig. 2.4D). Thus, our data suggests that both IKK subunits
play a role in regulating constitutive NF-κB activity in pancreatic cancer cells, but IKKα
may play a more significant role in Panc-1 cells.
IKK regulates pancreatic cancer cell growth and survival. To determine the
functional significance of NF-κB regulation by IKK in pancreatic cancer cells, growth and survival was measured following IKKβ inhibition (Compound A) or IKK RNA interference.
We showed that a higher concentration of Compound A (5μM) was required to significantly
57 reduce Panc-1 cell growth (Fig. 2.5A). However, MiaPaCa-2 cells were more sensitive to
increasing concentration of Compound A, as shown by significant loss of cell growth after 48
hour treatment at 1, 3, and 5μM (Fig. 2.5A). These results are consistent with our previous
observations of Compound A being more effective in suppressing constitutive NF-κB DNA
binding in MiaPaCa-2 cells (Fig. 2.4A). Cell growth was also measured in cells at 48, 72, and
96 hours post-transfection of IKKα and IKKβ siRNA. Furthermore, we used p65/RelA RNA
interference to test the dependency of NF-κB for cell growth. In Panc-1 cells, loss of either
IKK subunit or p65/RelA significantly reduced cell growth 48 hours post siRNA transfection
(Fig. 2.5B). However, knock-down of IKKα and p65/RelA was more effective in
suppressing cell growth at the 72 and 96 hour time-points (Fig. 2.5B). In MiaPaCa-2 cells,
knockdown of IKKα, IKKβ, and p65/RelA each resulted in a significant reduction in cell
growth 96 hours post siRNA transfection (Fig. 2.5B). We note that MiaPaCa-2 cell growth is
more sensitive to the loss of both IKK subunits, while Panc-1 cell growth is more dependent
on IKKα. While siRNA effects are not complete in their ability to knockdown individual
proteins, these experiments demonstrate that the IKK subunits and p65/RelA are involved in
growth/ survival of pancreatic cancer cells.
The induction of caspase-3 cleavage was also measured to determine whether the
reduction of cell growth following the disruption of IKK was due to apoptosis. We observe a dose-dependent increase in caspase-3 cleavage upon 24 hour treatment with Compound A in
Panc-1 cells (Fig. 2.5C). Similar results were obtained with MiaPaCa-2 cells (data not shown). Additionally, maximal caspase-3 cleavage was induced in Panc-1 cells following knock-down of both IKKα and IKKβ subunits (Fig. 2.5D). Furthermore, we observe a significant increase in caspase-3/7 activity following RNA interference against either IKKα
58 or IKKβ (Supplemental Fig. 2.4). Overall, our data suggest that differential NF-κB
regulation by IKK subunits correlates with cell growth and survival in Panc-1 and MiaPaCa-
2 cells.
GSK-3 is required for constitutive IKK activity. The mechanism by which GSK-3 regulates constitutive NF-κB signaling in pancreatic cancer is poorly understood. Our data thus far has shown that loss of GSK-3 isoforms and IKK subunits suppresses constitutive
NF-κB activity, cell growth and survival in Panc-1 and MiaPaCa-2 cells. To determine whether GSK-3 regulates constitutive IKK activity in pancreatic cancer cells, the effects of pharmacological GSK-3 inhibition on IKK-dependent IκBα phosphorylation was measured via an in vitro IKK kinase assay. Panc-1 cells were treated for 24 hours with GSK-3 inhibitor (AR-A014418) at 25 and 50μM. Endogenous IKK complexes were immunoprecipitated using an IKKα-specific antibody. Immunoprecipitates were incubated with GST-IκBα and an in vitro kinase reaction was performed. GSK-3 inhibition reduced phosphorylation of GST-IκBα in a dose-dependent manner (Fig. 2.6A). Moreover, the basal phosphorylation status of IκBα at serine 32/36 was measured in Panc-1 cells following the knock-down of both GSK-3 isoforms. Consistent with our in vitro kinase assay data, the loss of GSK-3 also resulted in a reduction of endogenous IκBα phosphorylation (Fig. 2.6B), and is consistent with an inhibition of IKK activity. Notably, these data provide the first evidence that GSK-3 regulates cancer cell-associated NF-κB by maintaining IKK activity (Fig. 2.7).
59 2.5 Discussion
GSK-3β has been previously reported to play an essential role in maintaining constitutive
NF-κB reporter activity and expression of NF-κB target genes in pancreatic cancer cells
(Ougolkov, Fernandez-Zapico et al. 2005). In this study, we show that both GSK-3 isoforms
function to regulate constitutive NF-κB activity in Panc-1 and MiaPaCa-2 cells. Although
both GSK-3 isoforms contribute to regulating NF-κB activity, GSK-3α was shown to be
primarily required for cell growth (Fig. 2.3B and Supplemental Fig. 2.2A). Moreover, the
suppression in cell growth following the loss GSK-3α may be due to increased apoptotic signaling, as shown by an induction of caspase-3 activity (Fig. 2.3D and Supplemental Fig.
2.3). We note that pharmacological inhibition of GSK-3 was more effective than GSK-3α
knock-down in suppressing Panc-1 cell growth (Fig. 2.3A and 2.3B). Although the GSK-3 inhibitor (AR-A014418) was not shown to affect a panel of closely related protein kinases
(Bhat, Xue et al. 2003), unknown off-target effects cannot be ruled out when interpreting
growth inhibition in pancreatic cancer cells. Importantly, we show that complete knock-
down of GSK-3 and significant growth suppression was not observed until later time-points
post siRNA transfection (Supplemental Fig. 2.2). These data may also explain why GSK-3α
siRNA was less effective in suppressing cell growth relative to GSK-3 pharmacologic
inhibition. Overall, our data suggest that GSK-3 isoforms are not functionally redundant in
regulating NF-κB activity and cell growth in pancreatic cancer cells.
IκB kinases play a central role in regulating NF-κB signal transduction. Therefore, efforts
have been made in utilizing IKK as a therapeutic target to block constitutive NF-κB activity.
In this report, we use an IKKβ-specific small molecule inhibitor (Compound A) that has been
shown to be effective in blocking constitutive (Duncan, Goetz et al. 2008) and inducible
60 (Ziegelbauer, Gantner et al. 2005) NF-κB activity in various cell lines. Our data shows that
Compound A is also efficient in inhibiting constitutive NF-κB activity (Fig. 2.4) and cell
proliferation (Fig. 2.5) in pancreatic cancer cells. In addition to IKKβ inhibition, we show
that IKKα is the predominant regulator of constitutive NF-κB activity and cell growth in
Panc-1 (Fig. 2.4, 2.5). Notably, higher concentrations of the IKKβ inhibitor (Compound A)
were required to diminish Panc-1 cell growth. These data raises the possibility that
Compound A may target IKKα at higher doses. Indeed, Comound A was shown to inhibit
recombinant IKKα activity at higher concentrations (Ki for ATP: 135nM) (Ziegelbauer,
Gantner et al. 2005). Overall, we show that the individual requirements for IKKα and IKKβ
for driving NF-κB activity vary between Panc-1 and MiaPaCa-2 cells. These data
underscore the heterogeneity between pancreatic carcinoma cell lines and prompts the need
to understand the variety of oncogenic mutations that potentiates NF-κB activity. Moreover,
our results emphasize the need for IKKα-specific small molecule inhibitors to target
constitutive NF-κB activity in select pancreatic carcinoma cell types.
The mechanism by which GSK-3 regulates constitutive NF-κB activity in pancreatic
cancer is not fully understood. Our data suggests that GSK-3 and IKK may function together
to regulate constitutive NF-κB activity. Previous reports have speculated on whether IKK is
required for GSK-3-dependent NF-κB activity. Analysis from our group and others provide
evidence that GSK-3β functions independent of the IKK complex during TNF-α-induced
NF-κB signaling (Hoeflich, Luo et al. 2000; Schwabe and Brenner 2002; Steinbrecher,
Wilson et al. 2005). Moreover, GSK-3 was also suggested to function independent of IKKβ
in pancreatic cancer cell lines(Ougolkov, Fernandez-Zapico et al. 2005). In this regard, our
61 current data demonstrates GSK-3-dependent regulation of constitutive IKK activity in certain
pancreatic cancer cell lines. We show that inhibition (AR-A014418) or knock-down of both
GSK-3 isoforms suppressed constitutive IKK activity in Panc-1 cells (Fig. 2.6). Notably, this
abrogation in constitutive IKK activity correlated with reduced NF-κB activity, cell growth,
and survival. Thus, we provide the first evidence that links GSK-3 and IKK in constitutive
NF-κB signaling in pancreatic cancer cells (Fig. 2.7). Further studies are needed to determine whether GSK-3 directly or indirectly regulates IKK activity.
Pancreatic cancer is a highly aggressive, metastatic disease known for its dismal mortality
rate. Despite current chemotherapeutic efforts to improve clinical prognosis, pancreatic
cancer still remains to be among the most drug-resistant tumors. Consequently, there is
growing interest on specifically targeting deregulated signaling pathways that drive the
molecular pathogenesis of pancreatic cancer. We emphasize in this report the critical roles
both GSK-3 and IKK play in maintaining constitutive NF-κB signaling. Collectively, our
data provide new insight into GSK-3-dependent NF-κB regulation, and further establishes
GSK-3 and IKK as potential therapeutic targets for pancreatic cancer.
62
Figure 2.1. GSK-3 is required for constitutive NF-κB DNA binding. EMSA was performed on Panc-1 and MiaPaCa-2 nuclear extracts using 32P-labeled NF-κB-specific probe. (A) Super-shift analysis was performed using antibodies against p65 and p50. Arrows indicate p65/50 and p50/p50 NF-κB complexes. Non-specific binding is specified by NS. (B) Cells were treated with vehicle control (DMSO) or GSK-3 inhibitors (AR-A014418 and SB216763) at 15 and 25μM for 24 hours. (C) Panc-1 cells were transiently transfected with 100nM siRNA targeted against GSK-3α, GSK-3β, combined GSK-3α/β, and non-targeting control (siCTRL) for 48 hours. Cytoplasmic extracts were harvested and separated by SDS- PAGE. Immunoblots were performed using the specified antibodies. (D) Nuclear extracts were harvested from Panc-1 and MiaPaCa-2 cells transfected with siRNA as described above and EMSA was performed.
63
Figure 2.2. GSK-3 regulates NF-κB transcriptional activity. (A) Panc-1 and MiaPaCa-2 cells were transiently transfected with 100nM siRNA targeted against GSK-3α, GSK-3β, combined GSK-3α/β, and non-targeting control (siCTRL) for 24 hours. Cells were subsequently transfected with 3X-NF-κB and renilla luciferase reporter constructs for 24 hours. Luciferase activity was measured in triplicate and indicated as % activity relative to siCTRL. Reporter activity from cells transfected with GSK-3 siRNA were all found to be significantly different relative to siCTRL (P < 0.05). (B) Panc-1 cells were transfected with siRNA as described above for 48 hours. RNA was harvested and expression of IκBα and Bcl-xL was analyzed by real-time PCR in triplicate. Astericks indicates statistical significance relative to siCTRL (P < 0.05). (C) Panc-1 and MiaPaCa-2 were transiently transfected with siRNA as described above for 48 hours. Whole cell extracts were harvested and separated by SDS-PAGE. Immunoblots were performed using the specified antibodies.
64
Figure 2.3. Blockade of GSK-3 suppresses cell proliferation. (A) Panc-1 cells were treated with DMSO or 5, 15, and 30μM of the GSK-3 inhibitor (AR-A014418) for 48, 72, and 96 hours. Cell growth was measured in triplicate at each time-point using a colormetric MTS tetrazolium assay. (B) Panc-1 cells were transiently transfected with siRNA targeted against GSK-3α, GSK-3β and siCTRL. Cell growth was measured as described above at 48, 72, and 96 hours post-transfection. Data was normalized to the initial cell density prior to GSK-3 inhibitor treatment or siRNA transfection respectively. Astericks indicates statistical significance relative to siCTRL (P < 0.05). (C) Panc-1 cells were treated with DMSO or 30μM of GSK-3 inhibitors (AR-A014418 and SB216764) for 24 and 48 hours. Whole-cell extracts were harvested and separated by SDS-PAGE. (D) Panc-1 cells were transiently transfected with 100nM siRNA targeted against GSK-3α, GSK-3β, combined GSK-3α/β, and siCTRL for 48 hours. Whole-cell extracts were harvested and separated by SDS-PAGE. Immunoblots were performed using the specified antibodies.
65
Figure 2.4. IKK is required for constitutive NF-κB activation. (A) Panc-1 and MiaPaCa- 2 cells were treated with DMSO or 0.5, 1, 3, and 5μM of IKKβ inhibitor (Compound A) for 24 hours. Nuclear extracts were harvested and EMSA was performed using 32P-labeled NF- κB-specific probe. Arrows indicate NF-κB complexes as determined by supershift analysis in Fig.1A. Non-specific binding is indicated with NS. (B) Panc-1 cells were transiently transfected with 100nM siRNA targeted against IKKα, IKKβ, combined IKKα/β, and siCTRL for 48 hours. Cytoplasmic extracts were harvested from cells transfected with siRNA as described above. Extracts were separated by SDS-PAGE and immunoblots were performed using the specified antibodies. (C) EMSA was performed on nuclear extracts harvested from cells transfected with siRNA as described above. (D) Panc-1 and MiaPaCa-2 cells were transfected with siRNA as described above for 24 hours. Cells were subsequently transfected with 3X-NF-κB and renilla luciferase reporter constructs for 24 hours. Luciferase activity was measured in triplicate and indicated as % activity relative to siCTRL. Reporter activity from cells transfected with IKK siRNA were all found to be significantly different relative to siCTRL.
66
Figure 2.5. Blockade of IKK suppresses cell proliferation. (A) Panc-1 and MiaPaCa-2 cells were treated with DMSO or 1, 3, and 5μM of IKKβ inhibitor (Compound A) for 48, 72, and 96 hours. Cell growth was measured in triplicate at each time-point using a colormetric MTS tetrazolium assay. (B) Panc-1 and MiaPaCa-2 cells were transiently transfected with 100nM siRNA targeted against IKKα, IKKβ, p65, and siCTRL. Cell growth was measured as described above at 48, 72, and 96 hours post-transfection. Data was normalized to the initial cell density prior to Compound A treatment or siRNA transfection respectively. Asterisks indicate statistical significance relative to DMSO or siCTRL (P < 0.05). (C) Panc-1 cells were treated with Compound A as described above for 24 hours. Whole-cell extracts were harvested and separated by SDS-PAGE. (D) Panc-1 cells were transiently transfected with 100nM siRNA targeted against IKKα, IKKβ, combined IKKα/β, and siCTRL. Whole- cell extracts were harvested, separated by SDS-PAGE, and immunoblotted using the specified antibodies.
67
Figure 2.6. GSK-3 inhibition suppresses constitutive IKK kinase activity. (A) Panc-1 cells were treated with DMSO or GSK-3 inhibitor (AR-A014418) at 25 and 50μM for 24 hours. As control, cells were treated with 10ng/ml TNF-α for 15min. Whole cells extracts were harvested. IKKα antibody was used to immunoprecipitate IKK complex. Kinase activity within immunocomplexes were assayed following incubation with wild-type GST- IκBα substrate in the presence of γ[32P]-ATP. Autoradiography was quantified and indicated as fold induction relative to DMSO. Immunoblots were performed against IKKα and GST to confirm equal loading. (B) Panc-1 cells were treated with 10ng/ml TNF-α for 15 minutes or transiently transfected with 100nM siRNA targeted against GSK-3α/β and siCTRL for 48 hours. Whole-cell extracts were harvested, separated by SDS-PAGE, and immunoblotted using the specified antibodies.
68
Figure 2.7. Model for GSK-3-dependent regulation of constitutive NF-κB activity in pancreatic cancer cells. GSK-3 regulates constitutive IKK phosphorylation (serine 176/ 180) and activity through an undefined mechanism (dashed arrow). IKK facilitates the phosphorylation (serine 32/ 36) and rapid proteosomal degradation of IκBα. Consequently, NF-κB (p65/ p50 heterodimer) is activated and regulates gene expression required for cell growth and survival of pancreatic cancer cells. GSK-3 inhibition (AR-A014418) and GSK-3 knock-down suppresses constitutive IKK activity. IKKβ inhibition (Compound A) and IKKα/β siRNA down-regulates pro-survival NF-κB signaling.
69
Supplemental Figure 2.1. GSK-3 regulates basal p65 DNA binding activity. Panc-1 and MiaPaCa-2 cells were transiently transfected with 100nM siRNA targeted against GSK-3α, GSK-3β, combined GSK-3α/β, and non-targeting control (siCTRL) for 48 hours. Nuclear extracts were harvested and p65 DNA binding activity was measured using an NF-κB DNA binding ELISA. Samples were measured at 450nm in triplicate and indicated as % siCTRL. Astericks indicates statistical significance relative to siCTRL (P < 0.05).
70
Supplemental Figure 2.2. Loss of GSK-3α attenuates cellular proliferation. (A) Panc-1 cells were plated in triplicate at 1 x 105 cells per well (six-well plate). Cells were transfected with 100nM siRNA targeted against GSK-3α, GSK-3β and non-targeting (siCTRL). Cells were harvested and total cell counts were performed at 48, 72, and 96 hours post- transfection. Astericks indicates statistical significance relative to siCTRL (P < 0.05). (B) Whole-cell extracts from each time point described above were harvested and separated by SDS-PAGE. Immunoblots were performed using antibodies against GSK-3α/β to confirm sufficient knock-down of GSK-3 isoforms at each time point post siRNA transfection.
71
Supplemental Figure 2.3. Loss of GSK-3α induces caspase-3/7 activity. Panc-1 cells were transfected with 100nM siRNA targeted against GSK-3α, GSK-3β, combined GSK- 3α/β, and non-targeting (siCTRL). Relative caspase-3/7 activity was measured in triplicate at 72 hours post siRNA transfection using a luminescent caspase-3/7 assay. Astericks indicates statistical significance relative to siCTRL (P < 0.05).
72
Supplemental Figure 2.4. Knock-down of IKK induces caspase-3/7 activity. Panc-1 cells were transfected with 100nM siRNA targeted against IKKα, IKKβ, combined IKKα/β, and non-targeting (siCTRL). Relative caspase-3/7 activity was measured in triplicate at 72 hours post siRNA transfection using a luminescent caspase-3/7 assay. Astericks indicates statistical significance relative to siCTRL (P < 0.05).
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76 CHAPTER III
GLYCOGEN SYNTHASE KINASE-3 DRIVES CONSTITUTIVE TAK1-IKK SIGNALING IN PANCREATIC CANCER CELLS
Portions of this chapter have been adapted from: Willie Wilson III and Al Baldwin.
Glycogen synthase kinase-3 drives constitutive TAK1-IKK signaling in pancreatic cancer cells. 2009. Manuscript in progress. 3.1 Abstract
Recent evidence has implicated glycogen synthase kinae-3 (GSK-3) in the regulation of
constitutive NF-κB activity in pancreatic cancer. However, the mechanism by which GSK-3
drives constitutive NF-κB activity remains elusive. We previously, reported that GSK-3
signals through IKK to facilitate downstream NF-κB signaling in pancreatic cancer cells.
Here, we expand this model and examine the functional link between GSK-3 and IKK. TGF-
β activated kinase 1 (TAK1) is a known regulator of IKK activity and has shown to be active
in human cancers. In this report, we provide initial evidence that TAK1 is constitutively
active in pancreatic cancer cells and plays a role in pro-survival NF-κB signaling. We show
that the phosphorylation of TAK1 at serine 412 can be suppressed following GSK-3
inhibition (AR-A014418) and GSK-3 knock-down in Panc-1 and MiaPaCa-2 cells.
Importantly, we observe a correlation between TAK phosphorylation at serine 412 and IKK activity. Overall, we propose that constitutive NF-κB activity in pancreatic cancer is driven by a novel GSK-3-TAK1-IKK signaling cascade. However, link between GSK-3 and TAK1 remains to be discovered. These data provide new insight into our understanding of constitutive NF-κB regulation in pancreatic cancer and presents TAK1 as a potential new therapeutic target for this disease.
78 3.2 Introduction
Pancreatic cancer is the most common malignancy of the pancreas and is ranked the 4th leading cause of cancer-related deaths in the United States (Jemal, Siegel et al. 2008).
Despite current therapeutic strategies, only 5% of patients diagnosed with this disease are expected to survive past 5 years (Jemal, Siegel et al. 2008). Advancements in pancreatic cancer chemotherapy have evolved into specifically targeting the many deregulated signaling pathways associated with disease progression (Berlin and Rothenberg 2003). Current research efforts have focused on understanding the complex mechanisms underlying constitutive activity of the transcription factor, NF-κB, in pancreatic cancer (Sarkar, Banerjee et al. 2006).
NF-κB represents a family of evolutionarily conserved, dimeric transcription factors consisting of RelA (p65), c-Rel, RelB, p50 (p105 precursor), or p52 (p100 precursor) subunits (Ghosh, May et al. 1998). The most abundant NF-κB complex consists of the p65/p50 heterodimer (Ghosh, May et al. 1998). NF-κB is sequestered in the cytoplasm of resting cells by interacting with inhibitory, IκB proteins. Classical NF-κB signal transduction is dependent on the activation of an IκB kinase (IKK) complex which consists of catalytic subunits (IKKα and IKKβ), and a regulatory subunit (IKKγ) (Zandi, Rothwarf et al. 1997). Following IKK complex activation, IκB undergoes phosphorylation and subsequent proteasomal degradation (Karin and Ben-Neriah 2000). Consequently, NF-κB can translocate to the nucleus and facilitate transcription of various target genes that regulate inflammation, proliferation, and apoptosis (Ghosh and Karin 2002).
Constitutive NF-κB activity has been linked to the progression of multiple human cancers and is the focus for targeted chemotherapies (Bharti and Aggarwal 2002; Aggarwal 2004).
79 NF-κB is constitutively active in 70% of pancreatic cancers and is known to facilitate cell
proliferation and survival in vitro (Zhang and Rigas 2006). Work from our lab and others
has demonstrated that constitutive NF-κB activity is dependent on IKK in pancreatic cancer
cell lines (Liptay, Weber et al. 2003; Li, Aggarwal et al. 2004; Wilson and Baldwin 2008).
The signaling events that drive constitutive IKK activity are not well understood. However,
we recently showed that glycogen synthase kinase-3 (GSK-3) may play a key regulatory role
in this pathway (Wilson and Baldwin 2008).
GSK-3 is a multifunctional serine/ threonine kinase that exists as two closely related
isoforms (GSK-3α and GSK-3β) (Woodgett 1990; Woodgett 1991). Numerous reports have
characterized GSK-3 as a key enzyme involved in diverse biological processes such as cell
cycle progression, differentiation, and apoptosis (Doble and Woodgett 2003). Moreover,
GSK-3 has recently been implicated as playing an oncogenic role in various human
malignancies including pancreatic cancer (Doble and Woodgett 2003; Ougolkov, Fernandez-
Zapico et al. 2005; Ougolkov, Bone et al. 2007; Rinnab, Schutz et al. 2008; Wang, Smith et
al. 2008; Zhou, Uddin et al. 2008). In fact, evidence suggests that GSK-3 regulates cell
growth and survival in pancreatic cancer cell lines by driving constitutive NF-κB activity
(Ougolkov, Fernandez-Zapico et al. 2005; Ougolkov, Fernandez-Zapico et al. 2006; Wilson and Baldwin 2008). Although the mechanism remains elusive, we recently proposed GSK-3 potentiates constitutive IKK and subsequent NF-κB activity in pancreatic cancer cells
through an unknown mechanism.
Transforming growth factor beta activated kinase 1 (TAK1) is a mitogen activated protein
kinase kinase kinase (MAP3K) that can initiate downstream NF-κB and MAPK signaling
cascades in response to cytokines (Ninomiya-Tsuji, Kishimoto et al. 1999). TAK1 activity is
80 dependent on its association with TAK1-binding proteins (TAB1, TAB2, TAB3) which
facilitate auto-phosphorylation (Thr184, Thr178, Ser192) within the kinase activation loop
(Kishimoto, Matsumoto et al. 2000; Yu, Ge et al. 2008) and recruitment to activated receptor complexes (Kanayama, Seth et al. 2004; Besse, Lamothe et al. 2007). Moreover, prostaglandin E2 was reported to enhance cytokine-induced TAK1 activity due to cyclic
AMP-dependent protein kinase A (PKA) phosphorylation of TAK1 at serine residue 412
(Kobayashi, Mizoguchi et al. 2005). Upon activation, TAK1 promotes the activation of p38 and JNK MAPK by phosphorylating MKK3/6 and MKK4 respectively (Raman, Chen et al.
2007). TAK1 also plays a central role in NF-κB activation through direct phosphorylation of
IKKβ (Wang, Deng et al. 2001).
Deregulated TAK1 signaling has been linked to constitutive IKK and NF-κB activity in
various human cancer cell lines (Jackson-Bernitsas, Ichikawa et al. 2007; Xie, Wang et al.
2009). A potential role for TAK1 in mediating constitutive NF-κB activity in pancreatic
cancer cells has yet to be characterized. In this report, we investigate the status of basal
TAK1 activity in a panel of pancreatic cancer cells and determine its role in driving
constitutive NF-κB signaling, cell proliferation, and survival. Moreover, we explore a
potential regulatory link between GSK-3 and TAK1, and propose that constitutive NF-κB
activity is driven by a unique GSK-3-TAK1-IKK signaling cascade. Collectively, these data
provide new insight in constitutive NF-κB regulation in pancreatic cancer, and establishes
GSK-3 and TAK1 as emerging therapeutic targets.
81 3.3 Materials and Methods
Cell culture and reagents. Panc-1 (CRL-1496), MiaPaCa-2 (CRL-1420), BXPC3 (CRL-
1687), and Capan-1 (HTB-79) were obtained from the American Type Culture Collection
(ATCC) (Manassas, VA). Cells were maintained in complete growth medium recommended
by ATCC and supplemented with 100 units/ml penicillin/ streptomycin. Human pancreatic
ductal epithelial cell line (HPDE6) was a generous gift from Dr. Channing Der (University of
North Carolina at Chapel Hill, Chapel Hill, NC) and was maintained in keratinocyte serum-
free growth medium. All cell culture reagents were obtained from Invitrogen (Carlsbad,
CA). The following antibodies were purchased from Cell Signaling (Beverly, MA):
phospho-IKK α/β (Ser176/180), phospho-MKK3/6 (Ser189/207), MKK6, phospho-p65
(Ser536), phospho-TAK1 (Ser412), phospho-TAK1 (Thr184/187), TAB1, and Flag-tag. The
following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA):
GSK-3α/β (SC-7291), TAK1 (SC-7162), p65 (SC-109), β-tubulin (SC-9104), and GAPDH
(SC-25778). IKKα clone 14A231 antibody was obtained from Upstate Biotechnology (Lake
Placid, NY). Flag-tag (M2) antibody was purchased from Sigma-Aldrich (St. Louis, MO).
PKA inhibitors (H-89 Dihydrochloride and Myristoylated PKI 14-22 amide) were purchased
from Cell Signaling (Beverly, MA) and EMD Chemicals (La Jolla, CA) respectively. GSK-3
inhibitor (AR-A014481) was purchased from Sigma-Aldrich (St. Louis, MO).
Expression plasmids. Flag-tagged, wild-type and dominant-negative (K63A) TAK1
plasmids were a generous gift from Dr. Derek Abbott (Abbott, Yang et al. 2007). TAK
(serine 412 to alanine) mutagenesis was performed on the wild-type TAK1 plasmid using the
QuikChange II Site-Directed Mutagenesis kit (La Jolla, CA) in accordance to manufacturer’s
instructions. Flag-tagged TAB1 plasmid was a generous gift from Dr. Jun Ninomiya-Tsuji
82 (North Carolina State University, Raleigh, NC). Transient transfections were performed overnight with Lipofectamine LTX (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions.
Small RNA interference. The following human siRNA (siGenome SMARTpool) was purchased from Dharmacon (Layfayette, CO) as a pool of four annealed double-stranded
RNA oligonucleotides: MAP3K7 (M-003790-06), RELA (M-003533-02), GSK-3α (M-
003009-01), GSK-3β (M-003010-03), and non-targeting control #3 (D001201-03).
Dharmafect 1 transfection reagent (Layfayette, CO) was used to transfect 100nM siRNA according to manufacturer’s instruction.
Western blot analysis and co-immunoprecipitation. Whole-cell lysates were prepared on ice using Mammalian Protein Extraction Reagent (M-PER) (Pierce Biotechnology,
Rockford, IL) supplemented with 1mM dithiothreitol (DTT), 1X protease inhibitor cocktail
(Roche Applied Science, Indianapolis, IN), and 1X phosphatase inhibitor cocktail 1 (Sigma-
Aldrich, St. Louis, MO). Protein concentrations were quantified by Bradford assay reagent
(Bio-Rad Laboratories, Hercules, CA) and analyzed by SDS-PAGE as previously described
(Wilson and Baldwin 2008). Co-immunoprecipitation was performed as previously described (Sakurai, Miyoshi et al. 1999).
NF-κB DNA binding ELISA. Nuclear extracts were harvested as previously described
(Steinbrecher, Wilson et al. 2005). Relative p65 DNA binding was quantified using the
TransAM NF-κB transcription factor assay kit (Active Motif, Carlsbad, CA) in accordance to manufacturer’s instructions. Samples were measured in triplicate at 450nm wavelength on a
Versamax Microplate Reader (Molecular Devices Corp., Sunnyvale, CA).
83 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium (MTS tetrazolium) cellular proliferation assay. Cells were transiently
transfected with siRNA as described above and cellular proliferation was measured at the
indicated times post-transfection using the CellTiter 96 Aqueous solution (MTS tetrazolium
compound) in accordance with the manufacturer’s instructions (Promega, Madison, WI).
Samples were measured in triplicate at 490nm wavelength on a Versamax Microplate Reader
(Molecular Devices Corp., Sunnyvale, CA).
Luminescent-based caspase-3/7 activity assay. Relative caspase-3/7 activity was
measured using the Caspase-Glo 3/7 assay (Promega, Madison, WI) as previously described
(Wilson and Baldwin 2008). Relative light units were measured in triplicate on an Lmax
Microplate Luminometer (Molecular Devices Corp., Sunnyvale, CA).
Dual-luciferase reporter assay. Cells were transiently transfected with wild-type or
mutant TAK1 plasmids as indicated above. In addition, cells were co-transfected with an
NF-κB-responsive luciferase reporter and a pRL-TK Renilla luciferase reporter (Promega,
Madison, WI) using Lipofectamine LTX (Invitrogen, Carlsbad, CA). Relative reporter activity was measured in triplicate using the Dual-Luciferase Assay System protocol
(Promega, Madison, WI) as previously described (Wilson and Baldwin 2008).
Non-radioactive PKA activity assay. PKA activity was measured using the Non-
radioactive Protein Kinase Assay Kit (EMD Chemicals, La Jolla, CA) according to
manufacturer’s instructions. In brief, cell lysates were mixed with kinase buffer (25mM
Tris-HCl, 5mM β-mercaptoethanol, 3mM MgCl2, 1mM EGTA, 0.5mM EDTA, and 0.1mM
ATP, pH 7.0) then incubated in a 96-well plate coated with PKA pseudosubstrate peptide. A biotinylated monoclonal antibody was used to detect PKA-dependent peptide
84 phosphorylation, and horseradish peroxidase conjugated streptavidin antibody was used to quantitate phosphorylation levels. Samples were measured in triplicate at 492 nm on a
Versamax Microplate Reader (Molecular Devices Corp., Sunnyvale, CA).
Statistical analysis. Statistical analysis of data was performed with Prism software
(GraphPad Software, Inc. San Diego, CA). An unpaired t test was used to evaluate differences between group means. P values less than 0.05 were considered to be statistically different.
3.4 Results
TAK1 is constitutively active in pancreatic cancer cells. The phosphorylation of TAK1 at threonine 184/187 (Yu, Ge et al. 2008) and serine 412 (Kobayashi, Mizoguchi et al. 2005) has been associated with TAK1 activity. To determine whether TAK1 is active in pancreatic cancer, we examined the status of TAK1 phosphorylation in a panel of pancreatic cancer cell lines (Panc-1, MiaPaCa-2, BXPC3, and CaPan1) by western blot analysis. As a control, we also measured TAK1 phosphorylation in an immortalized, human pancreatic ductal epithelial cell line (HPDE6). Relative to HPDE6 cells, we observed a significant increase in phosphorylation of TAK1 at serine 412. Moreover, TAK1 phosphorylation at threonine 184/
187 was also elevated, but to a lesser degree than serine 412 (Fig. 3.1). To confirm TAK1 activity, we also measured the phosphorylation of known TAK1 substrates (IKK and
MKK3/6). IKK phosphorylation (serine 176/180) was elevated in the pancreatic cancer cell lines with the highest levels in Panc-1 and CaPan-1 cells (Fig. 3.1). Notably, these data are consistent with previous reports of constitutive IKK and NF-κB activity in pancreatic cancer cell lines (Liptay, Weber et al. 2003; Li, Aggarwal et al. 2004). Similarly, we also detected increased levels of MKK3/6 (serine 189/207) phosphorylation in the pancreatic cancer cell
85 lines relative to HPDE6 cells (Fig. 3.1). Overall, these data provide initial evidence of
constitutive TAK1 activity in pancreatic cancer cell lines.
TAK1 depletion down-regulates pro-survival NF-κB activity in pancreatic cancer
cells. We previously reported that IKK mediates constitutive NF-κB activity in pancreatic
cancer cell lines (Panc-1 and MiaPaCa-2) (Wilson and Baldwin 2008). To determine
whether this activity is dependent on TAK1, we measured the effects of TAK1 depletion on
constitutive IKK substrate phosphorylation. Panc-1 and MiaPaCa-2 cells were transiently
transfected with TAK1-specific siRNA for 48 hours. TAK1 knock-down resulted in
significant depletion of TAK1 protein levels relative to non-targeting control (Fig. 3.2A).
We observe diminished phosphorylation of the IKK substrate (IκBα at serine 32/36)
following TAK1 depletion in Panc-1 and MiaPaCa-2 cells (Fig. 3.2A). The stabilization of
total IκBα levels following TAK1 knock-down also indicated reduced phosphorylation and
proteasomal degradation IκBα. IKK is also known to phosphorylate p65/RelA at serine 536
to promote NF-κB transcriptional activity at target gene promoters (Mattioli, Sebald et al.
2004). Similar to IκBα, TAK1 knock-down resulted in diminished p65 phosphorylation
relative to non-targeting control (Fig. 3.2A). Moreover, these results correlated with reduced
p65 DNA binding following TAK1 depletion (Fig. 3.2B). Taken together, these results
indicate that TAK1 plays a key role in regulating constitutive IKK and subsequent NF-κB
activity in pancreatic cancer cells.
To determine the functional significance of TAK1-mediated NF-κB activity, we examined
the effect of TAK1 depletion on pancreatic cancer cell growth/survival. MiaPaCa-2 cells
were transiently transfected with TAK1-specific siRNA and cell proliferation was measured at 48, 72, and 96 hours post-transfection. TAK1 depletion significantly reduced cell
86 proliferation relative to non-targeting control throughout the time-course (Fig. 3.2C).
Moreover, these results were similar to the growth suppression observed following the
knock-down of the NF-κB subunit, p65/RelA (Fig. 3.2C). Next, we determined whether this abrogated cell growth correlated with the induction of apoptosis as measured by caspase-3/ 7
activity. MiaPaCa-2 cells were transiently transfected with TAK1 and p65-specific siRNA
for 48 hours and caspase-3/7 activity was measured. We observed an increase in caspase-3/
7 activity following knock-down of either TAK1 or p65 (Fig. 3.2D). Thus, these data
provide evidence that TAK1 is active in pancreatic cancer cells and mediates constitutive
pro-survival NF-κB signaling.
Blockade of GSK-3 suppresses constitutive phosphorylation of TAK1 at serine 412.
We proposed that GSK-3 drives constitutive IKK and NF-κB activity in pancreatic cancer
cell lines through an unknown mechanism (Wilson and Baldwin 2008). To determine
whether GSK-3 potentiates TAK1-dependent NF-κB signaling, we measured the effect of
GSK-3 inhibition (AR-A014418) on TAK1 phosphorylation. Panc-1 and MiaPaCa-2 cells
were treated for 24 hours with AR-A014418 and TAK1 phosphorylation at serine 412 and
threonine 184/187 was measured by western blot analysis. Interestingly, we observed a
reduction in serine 412 phosphorylation in both cell lines with the maximum effect seen in
Panc-1 cells (Fig. 3.3A). Notably, TAK1 phosphorylation at threonine 184/ 187 was not
effected by GSK-3 inhibition. Next, we examined whether the reduced serine 412
phosphorylation of TAK1 correlated with decrease substrate phosphorylation of IKK and
p65. Consistent with our previous data (Wilson and Baldwin 2008), GSK-3 inhibition also
suppressed IKK and p65 phosphorylation in Panc-1 and MiaPaCa-2 cells (Fig. 3.3A).
87 AR-A014481 targets the inhibition of GSK-3α and GSK-3β, but potential off-target effects from this inhibitor cannot be ruled out. Therefore, we measured the effects of GSK-
3α/β depletion on TAK1 phosphorylation and subsequent NF-κB activity. Panc-1 and
MiaPaca-2 cells were transiently transfected with GSK-3α/β-specific siRNA for 48 hours and significant reduction in GSK-3 protein levels were observed relative to non-targeting siRNA control (Fig. 3.3B). Consistent with GSK-3 inhibition, GSK-3α/β knock-down resulted in the downregulation of TAK1 phosphorylation at serine 412 and not threonine 184/
187 (Fig. 3.3B). Furthermore, the reduced TAK1 phosphorylation at serine 412 correlated with decreased phosphorylation of IKK and p65 in both cell lines (Fig. 3.3B). Overall, these data provide initial evidence that links GSK-3 to constitutive NF-κB signaling in pancreatic cancer cells via phosphorylation of TAK1 at serine 412.
TAK1 phosphorylation at serine 412 is necessary to induce IKK phosphorylation.
The phosphorylation of TAK1 at serine 412 by PKA was reported to enhance cytokine- induced NF-κB activity in RAW264.7 macrophage cells (Kobayashi, Mizoguchi et al. 2005).
To determine whether this TAK1 phosphorylation is also required to enhance NF-κB activity in pancreatic cancer cells, we utilized ectopic expression of a serine 412 to alanine TAK1 point mutant. To confirm TAK1 mutagenesis, Panc-1 cells were transiently transfected with flag-tagged, wild-type and mutant TAK1 (S412A), then stimulated with forskolin to induced
PKA-dependent TAK1 phosphorylation. Following flag-tagged TAK1 immunoprecipitation, we confirmed that TAK1 (S412A) was deficient in basal and inducible phosphorylation relative to wild-type TAK1 (Fig. 3.4A). Since ectopic expression of TAK1 alone is not sufficient to induced NF-κB activity, TAK1 must be co-expressed with TAK1 binding protein (TAB1) (Kishimoto, Matsumoto et al. 2000). To determine whether S412 TAK1
88 phosphorylation is required to induce NF-κB signaling, we measured basal IKK
phosphorylation upon TAK1 and TAB1 co-expression in Panc-1 cells. Co-expression of
TAB1 with wild-type TAK1 significantly induced IKK phosphorylation relative to vector
control (Fig. 3.4B). In contrast, TAK1 (S412A) failed to induce IKK phosphorylation (Fig.
3.4B). Furthermore, this result was consistent with the expression of kinase dead TAK1
(K63A) mutant and suggests serine 412 phosphorylation promotes TAK1 catalytic activity
against IKK (Fig. 3.4B). Next, we measured the ability of TAK1 (S412A) to induce an NF-
κB luciferase reporter upon TAB1 co-expression. Consistent with IKK phosphorylation,
wild-type TAK1 expression significantly induced NF-κB reporter activity while kinase dead
TAK1 (K63A) was deficient in this response (Fig. 3.4C). However, the TAK1 (S412A)
mutant retained the ability to induce NF-κB reporter activity, but the level of activity was
significantly less than wild-type TAK1 (Fig. 3.4C). Thus, these data suggests that TAK1
phosphorylation at serine 412 plays a role in promoting IKK phosphorylation, but is only
partially required to induce NF-κB activity.
TAK1 phosphorylation at serine 412 is PKA-dependent. Cyclic-AMP-dependent PKA
was previously reported to be constitutively active in pancreatic cancer cells (Farrow,
Rychahou et al. 2003). Therefore, we investigated whether constitutive TAK1 (S412) and
IKK phosphorylation was dependent on PKA activity. Panc-1 cells were treated for 30
minutes with increasing concentrations (1, 15, 30 μM) of forskolin to measure the ability of
PKA to enhance basal TAK1 and IKK phosphorylation. Forskolin treatment resulted in a
dose-dependent increase in PKA activity relative to vehicle control (Fig. 3.5A) Moreover,
the increase in PKA activity following forskolin treatment correlated with enhanced TAK1 and IKK phosphorylation (Fig. 3.5B). Next, we examined whether constitutive TAK1 and
89 IKK phosphorylation was maintained by PKA activity in pancreatic cancer cells. Panc-1 cells were treated with the increasing concentrations of the PKA inhibitor (H89) for 24 hours.
Consistent with a previous report (Farrow, Rychahou et al. 2003), H89 treatment resulted in a dose-dependent reduction of basal PKA activity (Fig. 3.5C). In addition, we observed decreased phosphorylation of TAK and IKK following H89 treatment with the maximum effect seen at 30μM (Fig. 3.5D). However, the minor decrease in PKA activity following
1μM H89 did not correlated with the dramatic loss of TAK1 phosphorylation seen at the same dose. Potential off-target effects of H89-mediated PKA inhibition could explain these results (see discussion). Overall, these data confirms PKA as a TAK1 (S412) kinase and suggests a role for PKA in regulating TAK1 and subsequent IKK activity in pancreatic cancer cells.
GSK-3 is not required for forskolin-induced TAK1 phosphorylation at serine 412.
Our data suggests that GSK-3 promotes constitutive IKK and NF-κB activity through the phosphorylation of TAK1 at serine 412 (Fig. 3.3). However, the link between GSK-3 and
TAK1 is unclear since the TAK1 (S412) motif (NGQPRRRSIQDLTVT) does not fit the consensus substrate motif of GSK-3 (S/T-XXX-S/T). Therefore, we investigated whether
GSK-3 indirectly regulates basal and inducible TAK1 phosphorylation through PKA. Panc-1 cells were pre-treated with the GSK-3 inhibitor (AR-A014418) or vehicle control (DMSO) for 24 hours, and then treated with increasing concentration of forskolin for 30 minutes.
GSK-3 inhibition decreased overall levels of TAK1 (S412) phosphorylation relative to vehicle control (Fig.3.6A). However, forskolin treatment was still able to enhance TAK1 phosphorylation in a dose-dependent manner following GSK-3 inhibition (Fig. 3.6A). In contrast, forskolin-induced IKK phosphorylation was attenuated following treatment with
90 AR-A014418 (Fig. 3.6A). Next, we examined whether GSK-3 inhibition could suppress basal or forskolin-induced PKA activity in Panc-1 cells. We observe no significant change in basal PKA activity following GSK-3 inhibition (Fig. 3.6B). In addition, pre-treatment with
AR-A014418 slightly enhanced forskolin-induced PKA activity relative to vehicle control
(Fig. 3.6B). Collectively, these data suggests a model for forskolin-induced and constitutive
TAK1 (S412) phosphorylation (Fig.3.7): (1) PKA-dependent TAK1 phosphorylation in response to forskolin and (2) GSK-3-dependent basal TAK1 phosphorylation that does not require PKA (Fig. 3.7). We propose that constitutive NF-κB activity in pancreatic cancer cells is driven by a novel GSK-3-TAK1-IKK signaling cascade (Fig. 3.7). However, the regulatory link between GSK-3 and TAK1 (S412) phosphorylation remains to be discovered.
3.5 Discussion
The molecular mechanism underlying constitutive NF-κB activity in pancreatic cancer is complex and utilizes crosstalk from various pathways. Current research efforts have focused on expanding our knowledge of these pathways to identify novel therapeutic targets (Zhang and Rigas 2006). GSK-3 has been implicated in regulating constitutive NF-κB activity in pancreatic cancer (Ougolkov, Fernandez-Zapico et al. 2005; Ougolkov, Fernandez-Zapico et al. 2006; Wilson and Baldwin 2008). Moreover, this multi-tasking kinase has emerged over the years as a promising therapeutic target for pancreatic cancer and many other human malignancies (Ougolkov, Bone et al. 2007; Rinnab, Schutz et al. 2008; Wang, Smith et al.
2008; Zhou, Uddin et al. 2008). Nevertheless, the mechanism by which GSK-3 drives constitutive NF-κB activity in pancreatic cancer remains elusive. We previously reported the
GSK-3 signals through IKK to mediate constitutive NF-κB signaling in pancreatic cancer cells (Wilson and Baldwin 2008). In this report, we provide the first evidence that TAK1 is
91 constitutively active pancreatic cancer cell lines (Fig. 3.1) and propose that GSK-3 and IKK are functionally linked through the phosphorylation of TAK1 at serine 412 (Fig.3.7).
The phosphorylation of TAK1 at serine 412 was initially reported to enhance RANKL- induced NF-κB activity and osteoclast differentiation in response to prostaglandin E2
(PGE2) (Kobayashi, Mizoguchi et al. 2005). This novel phosphorylation on TAK1 was shown to be driven by cyclic AMP-dependent activation of PKA (Kobayashi, Mizoguchi et al. 2005). We demonstrated that TAK1 (S412) phosphorylation is significantly elevated in pancreatic cancer cells and may be responsible for driving constitutive NF-κB activity (Fig.
3.1). Co-expression of TAK1 (S412A) mutant with TAB1 failed to enhance basal levels of
IKK phosphorylation relative to wild-type TAK1. This data is consistent with a previous report that showed TAK1 (S412A) expression attenuated RANKL, LPS, and TNF-α-induced
IκBα degradation (Kobayashi, Mizoguchi et al. 2005). Despite being defective in inducing
IKK phosphorylation, TAK1 (S412A) mutant retained partial ability to induced NF-κB luciferase activity. This result would suggest that TAK (S412A) mutant can signal to NF-κB through a mechanism that is not dependent on IKK activity. We note that the TAK1 (S412) phosphorylation site resides outside the kinase domain and within the C-terminal TAB2/3 binding domain of TAK1. TAB2 and TAB3 associate with TAK1 and promotes its recruitment to K63-linked polyubiquitinated signaling adapters in response to stimuli
(Kanayama, Seth et al. 2004; Besse, Lamothe et al. 2007). Therefore, TAK1 (S412) phosphorylation could mediate TAB2/3 binding and subsequent TAK signaling. However, evidence showed that enhanced TAK1 (S412) phosphorylation by prostaglandin E2 failed to change TAK1-TAB2/3 association in response to RANKL (Kobayashi, Mizoguchi et al.
2005). Our knowledge of TAK (S412) phosphorylation is limited and further studies are
92 needed to fully elucidate its role in mediating constitutive TAK1 and NF-κB activity in pancreatic cancer cells.
In this study, we explored the potential mechanisms that drive TAK (S412) phosphorylation and subsequent downstream activation of IKK in pancreatic cancer cells.
Currently, PKA is the only known kinase to phosphorylate TAK1 at serine 412 (Kobayashi,
Mizoguchi et al. 2005). Our data also suggest that PKA can function as a TAK (S412) kinase in response to forskolin treatment (Fig. 3.5). Interestingly, there is also evidence that links PKA activity to pancreatic cancer. The pancreatic cancer cell line (JF-305) was shown to secrete prostaglandin E2 which facilitated PKA-dependent production of cyclooxygenase-
2 (COX2), as well as, cell proliferation, and survival (Wu, Yi et al. 2005). Moreover, the pharmacological PKA inhibitor (H89) was shown to suppress cell growth and induce apoptosis in Panc-1 and MiaPaCa-2 cells (Farrow, Rychahou et al. 2003). Our data indicates that H89-mediated PKA inhibition also results in decreased TAK1 (S412) and IKK phosphorylation in Panc-1 cells (Fig. 3.5). We note that the degree of H89-mediated PKA inhibition was not proportional to the levels of TAK1 (S412) and IKK phosphorylation (Fig.
3.5). Although H89 is an effective PKA inhibitor, several off-target effects have been associated with this compound (Lochner and Moolman 2006). For example, H89 has been shown to target PKC, PKG, S6K, and AMPK (Lochner and Moolman 2006). Therefore, these off-targets need to be taken into consideration when measuring H89-mediated suppression of TAK1 (S412) and IKK phosphorylation. Further studies are needed to rule out these H89 off-targets as potential TAK1 (S412) kinases. Moreover, the use of alternative
PKA inhibitors is necessary in establishing the role of PKA in regulating constitutive TAK1
(S412) and IKK phosphorylation in pancreatic cancer cells.
93 Our previous work implicated GSK-3 in regulating constitutive IKK activity and pro-
survival NF-κB signaling in pancreatic caner cells (Wilson and Baldwin 2008). Here, we
discovered that GSK-3 may be linked to IKK through the phosphorylation of TAK1 at serine
412 (Fig. 3.3). Thus, we proposed that constitutive NF-κB activity in pancreatic cancer cells
is driven by a novel GSK-3-TAK1-IKK signaling cascade. We note that the TAK (S412)
phosphorylation site (NGQPRRRSIQDLTVT) does not match the consensus GSK-3
substrate recognition motif (S/T-XXX-S/T). Therefore, we investigated indirect mechanisms
of GSK-3-dependent TAK1 phosphorylation. Since PKA plays a key role in TAK1 (S412)
phosphorylation (Kobayashi, Mizoguchi et al. 2005), we asked whether GSK-3 was required
for PKA-dependent phosphorylation of TAK1 (S412) and IKK phosphorylation. Our data
provides a model for both forskolin-induced and constitutive regulation of TAK1 (S412)
phosphorylation in pancreatic cancers cells (Fig. 3.7). Indeed, PKA prompts the induction of
TAK1 (S412) in response to forskolin. However, we also identified a novel mechanism by
which GSK-3 drives constitutive levels of TAK1 (S412) and IKK phosphorylation
independent of PKA. Additional studies are needed to discover the missing link between
GSK-3 and TAK1. These efforts will be dependent on finding alternative TAK1 (S412)
kinases and elucidates their regulation by GSK-3. As indicated above, the off-target kinases
of H89 may serve as potential candidates for identifying alternative TAK1 (S412) kinases.
Overall, our data underscores the complexity underlying TAK1-mediated NF-κB activity in pancreatic cancer cells and emphasizes the need to explore alternative mechanisms of
GSK-3-dependent TAK1 (S412) phosphorylation. Moreover, this report provides additional evidence that implicates GSK-3 and TAK1 as emerging therapeutic targets in pancreatic cancer.
94
Figure 3.1. TAK1 is constitutively active in pancreatic cancer cells. Whole cell lysates were harvested from human pancreatic cancer cell line (Panc-1, MiaPaCa-2, BXPC3, and CaPan-1) and an immortalized human pancreatic ductal epithelial cell line (HPDE6). Western blot analysis was performed with the indicated antibodies. The phosphorylation of TAK1 (Ser412 and Thr184/ 187), MKK3/6 (Ser189/207), and IKKα/β (Ser176/180) are indicative of TAK1 activity.
95
Figure 3.2. TAK1 mediates pro-survival NF-κB activity in pancreatic cancer cells. (A) Panc-1 and MiaPaCa-2 cells were transiently transfected with non-targeting (siCTRL) or TAK1 siRNA for 48 hours. Whole cell lysate were harvested and western blot analysis was performed. (B) Panc-1 and MiaPaCa-2 cells were transfected with siRNA as indicated above and nuclear extracts were harvested. An NF-κB (p65) DNA binding ELISA was performed to measure relative p65 activity. Samples were measured in triplicate and normalized to siCTRL for each cell line. Asterisk indicates statistical difference relative to siCTRL (P < 0.05). (C) MiaPaCa-2 cells were transfected with siRNA as indicated above and cell viability was measured at the indicated times post-transfection. Samples were measured in triplicate and normalized to untransfected cells. Asterisk indicates statistical difference relative to siCTRL at each time point (P < 0.05). (D) MiaPaCa-2 cells were transfected with siRNA for 48 hours. Apoptosis was measured using a luminescent-based caspase-3/7 assay. Samples were measured in triplicate and normalized to siCTRL. Asterisk indicates statistical difference relative to siCTRL (P < 0.05).
96
Figure 3.3. Blockade of GSK-3 suppresses constitutive phosphorylation of TAK1 at serine 412. (A) Panc-1 and MiaPaCa-2 cells were treated for 24 hours with 25μM GSK-3 inhibitor (AR-A014481), whole cell lysates were harvested, and western blot analysis was performed with the indicated antibodies. (B) Panc-1 and MiaPaCa-2 cells were transiently transfected with non-targeting control (siCTRL) or siRNA targeting GSK-3α and GSK-3β (siGSK-3α/β) for 48 hours. Whole cell lysates were harvested and western blot analysis was performed as indicated above.
97
Figure 3.4. TAK1 phosphorylation at serine 412 is necessary to induce IKK phosphorylation. (A) Panc-1 cells were transiently transfected with flagged wild-type or mutant (S412A) TAK1 for 24 hours. Cells were treated with 30μM Forskolin or vehicle control (DMSO). Whole cell extracts were harvested. Samples were immunoprecipitated with flag antibody or IgG control then analyzed by western blot with the indicated antibodies. (B) Panc-1 cells were transiently co-transfected with TAB1 and either vector control, wild-type TAK1, kinase dead TAK1 (K63A), or mutant TAK1 (S412A). Whole cell lysates were harvested and western blot analysis was performed. (C) Panc-1 cells were co- transfected as indicated above along with 3X NF-κB luciferase and renilla reporter. NF-κB reporter activity was measured in triplicate and indicated as fold-induction relative to vector control. Asterisk indicates statistical difference relative to wild-type TAK1 induction (P < 0.05).
98
Figure 3.5. TAK1 phosphorylation at serine 412 is PKA-dependent. (A) Panc-1 cells were treated with vehicle control (DMSO) or the increasing concentrations (1μM, 15μM, and 30μM) of forskolin for 24 hours. Whole cell lysates were harvested and PKA activity was measured using a non-radioactive PKA ELISA assay. Samples were measured in triplicate and normalized to DMSO. (B) Whole cell lysates from (A) were analyzed by western blot with indicated antibodies. (C) Panc-1 cells were treated with vehicle control (DMSO) or the increasing concentrations (1μM, 15μM, and 30μM) of PKA inhibitor (H89) for 24 hours. (C) PKA activity (D) western blot analysis was performed as indicated above. Asterisk indicates statistical difference relative to DMSO (P < 0.05).
99 Figure 3.6. GSK-3 is not required for forskolin-induced TAK1 phosphorylation at serine 412. (A) Panc-1 cells were pre-treated with vehicle control or 30μM GSK-3 inhibitor (AR-A014418) for 24 hours. Cells were treated with increasing concentrations (1μM, 15μM, and 30μM) of forskolin for 30 minutes, whole cell lysates were harvested, and western blot analysis was performed with the indicated antibodies. (B) PKA activity was measured in the whole cell lysates from (A) using a non-radioactive PKA ELISA assay. Samples were measured in triplicate and normalized to DMSO. Asterisk indicates statistical difference relative to DMSO in each group (P < 0.05).
100
Figure 3.7. Model for inducible and constitutive TAK (S412) phosphorylation in pancreatic cancer cells. We propose that the mechanism by which GSK-3 regulates constitutive TAK (S412) phosphorylation in pancreatic cancer is distinct from PKA-induced TAK1 phosphorylation in response to forskolin. GSK-3 drives TAK1 activity through indirect (dashed arrow) regulation of TAK1 phosphorylation at serine 412. TAK1 activates downstream IKK phosphorylation at serine 176/180, and subsequent IκBα phosphorylation (serine 32/36) and degradation. As a result, pro-survival NF-κB is activated. Forskolin increases intercellular levels of cyclic AMP and facilitates PKA-dependent TAK1 (S412) phosphorylation independent of GSK-3.
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105 CHAPTER IV
CONCLUSIONS & FUTURE DIRECTIONS
4.1 Conclusions and future directions
For more than a decade, the link between GSK-3 and cancer has been dominated by the
paradigm of GSK-3 functioning as a tumor suppressor (Lustig and Behrens 2003). This
paradigm is based on the Wnt/β-catenin pathway in human colorectal cancer where GSK-3
suppresses the oncogenic affect of β-catenin (Shakoori, Ougolkov et al. 2005). Recently, this
paradigm has been challenged by several reports that implicate a potential oncogenic role for
GSK-3 in pancreatic cancer (Ougolkov, Fernandez-Zapico et al. 2005; Wilson and Baldwin
2008), chronic lymphocytic B-cells (Ougolkov, Bone et al. 2007), prostate cancer (Rinnab,
Schutz et al. 2008), myeloid-lymphoid leukemia (MLL)(Wang, Smith et al. 2008), and
multiple myeloma (Zhou, Uddin et al. 2008). Indeed, GSK-3 is now emerging as a therapeutic target for various human malignancies in part for its role in regulating
constitutive NF-κB activity (Ougolkov and Billadeau 2006). Thus, there is a confounding
question centered on whether GSK-3 is an oncogene or tumor suppressor, and what adverse
affects GSK-3 inhibitors will have as a cancer therapy in the clinics. The fact of the matter
remains that cancer is a complex, heterogeneous disease. Therefore, the role GSK-3 plays in
cancer may be dependent on cancer cell type, tumor microenvironment, genetic background,
etc.
The previous chapters underscore the oncogenic role GSK-3 plays in pancreatic cancer.
Consistent with previous reports (Ougolkov, Fernandez-Zapico et al. 2005; Ougolkov,
Fernandez-Zapico et al. 2006), our data indicates that GSK-3 drives constitutive, pro-survival
NF-κB activity in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2). The primary focus of this project was to characterize GSK-3-dependent regulation of constitutive NF-κB
activity. Importantly, we provide initial evidence that GSK-3 facilitates pro-survival NF-κB
107 signaling by maintaining constitutive IKK activity. Although the link between GSK-3 and
IKK is not established, we explored a potential mechanism that utilizes a known regulator of
IKK, TAK1. Our data suggests that TAK1 is constitutively active in pancreatic cancer cells and plays a role in maintaining NF-κB signaling. Importantly, we hypothesize that GSK-3 may be linked to constitutive IKK and NF-κB through the novel phosphorylation of TAK1 at serine 412. However, further evidence is needed to establish that constitutive NF-κB activity in pancreatic cancer can be mediated through this GSK-3-TAK-IKK signaling cascade. The following questions should be addressed to support this model: (1) Is TAK1 phosphorylation at serine 412 essential for constitutive NF-κB activity in pancreatic cancer cells? (2) How does phosphorylation of TAK1 at serine 412 modulate TAK1 signaling? (3) What is the link between GSK-3 and constitutive TAK phosphorylation at serine 412?
(1) Is TAK1 phosphorylation at serine 412 essential for constitutive NF-κB activity in pancreatic cancer cells? TAK1 (S412) phosphorylation was originally discovered to enhance cytokine-induced NF-κB activity in response to prostaglandin E2 (Kobayashi,
Mizoguchi et al. 2005). However, the requirement for TAK1 (S412) phosphorylation in maintaining constitutive NF-κB activity in pancreatic cancer cells remains elusive. The experiments presented in Figure 3.4 utilized TAK1-TAB1 co-expression to investigate the ability of TAK (S412A) mutant to induce IKK and NF-κB activity. Although the TAK
(S412A) mutant was deficient in inducing IKK phosphorylation, it still retained the ability to induce NF-κB reporter activity. To explain this paradox, further experiments are needed to determine if TAK1-induced IKK is required for NF-κB activity in pancreatic cancer cells.
Other read-outs of NF-κB activity (i.e. NF-κB target gene expression, NF-κB DNA binding, and NF-κB nuclear localization) should be explored to fully ascertain the effect of TAK1
108 (S412A) mutation on NF-κB signaling. When interpreting these data, we must take into
consideration that other oncogenic factors (i.e. KRAS and AKT) may be signaling to IKK
and NF-κB through a TAK1-independent mechanism. We must also take into account that
ectopic expression of TAK1 creates an artificial system of NF-κB regulation that may not
recapitulate how endogenous levels of TAK1 regulate constitutive NF-κB.
(2) How does phosphorylation of TAK1 as serine 412 modulate TAK1 signaling?
The phosphorylation of TAK1 at serine 412 resides outside the catalytic domain and suggests
that this phosphorylation event does not control overall TAK1 kinase activity. Therefore,
additional experiments are needed to fully understand how TAK (S412) phosphorylation
controls TAK1 signaling. Serine 412 resides in the C-terminal, TAB2/3 binding domains of
TAK1. The function of TAB2/3 is to promote the recruitment of TAK1 to K63-linked
polyubiquitinated signaling adapters (i.e. TRAF6 and RIP1). Thus, TAK1 (S412)
phosphorylation could facilitate TAB2/3 binding and subsequent recruitment to these
signaling adapters in pancreatic cancer cells. To investigate this model, we must first
determine whether TAK (S412A) mutant has a decreased affinity for TAB2/3. Next, we
should ascertain the status of K63-linked polyubiquitination on TRAF6 or RIP1 and
determine whether endogenous TAK1 can associate with these signaling adapters.
(3) What is the link between GSK-3 and constitutive TAK phosphorylation at serine
412 regulated? We provide evidence that GSK-3 inhibition and knock-down correlate with the suppression of constitutive TAK1 (S412) phosphorylation in pancreatic cancer cells (Fig.
3.3). However, the link between GSK-3 and TAK1 requires further elucidation. We hypothesized that GSK-3 indirectly regulates TAK1 (S412) phosphorylation. The investigation of this regulatory mechanism is dependent on identifying potential TAK1
109 (S412) kinases. PKA was discovered to induce TAK1 (S412) phosphorylation in response to
prostaglandin E2 in RAW264.7 macrophage cells (Kobayashi, Mizoguchi et al. 2005).
However, our data suggest that GSK-3 may control constitutive TAK (S412) phosphorylation
in pancreatic cancer cells through a PKA-independent mechanism. In Figure 3.5, the PKA
inhibitor (H89) was used to investigate the role of PKA in regulating constitutive TAK1
(S412) phosphorylation in pancreatic cancer cells. Due to the off-target effects of H89,
questions were raised about alternative kinases that can phosphorylate TAK1. Additional
experiments should explore the off-target effect of H89 and determine whether they could be
regulated by GSK-3. For example, protein kinase C (PKC) represents a potential alternative
kinase that can phosphorylate TAK1 at serine 412. Constitutive PKC activity has also been
implicated in pancreatic cancer and is considered a therapeutic target (El-Rayes, Ali et al.
2008). Future experimentation should utilize selective PKC inhibitors to establish the role of
this kinase in regulating constitutive TAK1 (S412) phosphorylation and downstream NF-κB
signaling. Importantly, these studies should determine whether the blockade of GSK-3
affects PKC activity.
In addition to these immediate questions, this project also raises several fundamental
questions regarding GSK-3-dependent NF-κB regulation. (1) How is GSK-3 deregulated in
pancreatic cancer? (2) What is the normal physiological relationship between GSK-3 and
TAK1 under inducible NF-κB conditions? (3) What role do nuclear GSK-3 and IKKα play
in driving constitutive NF-κB activity in pancreatic cancer cells? (4) Is the activity of GSK-
3 and TAK1 required for pancreatic cancer progression in vivo?
(1) How is GSK-3 deregulated in pancreatic cancer? One of the main questions
underlying oncogenic GSK-3 is determining how this kinase becomes deregulated in cancer.
110 Currently, there have been no cancer-related mutations associated with GSK-3. Therefore,
GSK-3 may be deregulated by aberrant upstream signaling pathways. GSK-3 is a constitutively active kinase that is primarily known to be negatively regulated through phosphorylation at serine 9 (GSK-3β) and 21 (GSK-3α) (Doble and Woodgett 2003). We observe basal phosphorylation of these inhibitory markers in pancreatic cancer cells (data not shown). However, work from our lab and others suggest GSK-3 still retains the ability to activate NF-κB. This evidence indicates that GSK-3-dependent regulation of constitutive
NF-κB in pancreatic cancer is independent of inhibitory serine 9/21 phosphorylation.
Importantly, we also observe that blockade of GSK-3 activity does not lead to the stabilization of β-catenin in pancreatic cancer cells (data not shown). This may be due to constitutive activation of the Wnt pathway which sustains elevated levels of β-catenin. In fact, it was recently reported that pancreatic cancers have elevated expression of ataxia- telangiectasia group D complementing gene (ATDC) (Wang, Heidt et al. 2009). ATDC interacts with the negative regulator of GSK-3 (Disheveled-2) and drives β-catenin stabilization and oncogenesis (Wang, Heidt et al. 2009). Thus, there may be pools of GSK-3 existing outside of the β-catenin “destruction complex” that promotes constitutive NF-κB.
Nevertheless, further studies are needed to fully elucidate the context of GSK-3 deregulation in pancreatic cancer and its relationship with NF-κB pathway.
(2) What is the normal physiological relationship between GSK-3 and TAK1 under inducible NF-κB conditions? NF-κB activation is a tightly regulated process that can be triggered by a variety of inflammatory stimuli. We hypothesize in this project that NF-κB activity is driven by a novel GSK-3-TAK-IKK signaling cascade. In order to broaden our understanding of this signaling event, it is important determine how this process is normally
111 regulated under physiological conditions. Chapter 1 reviewed how GSK-3 and TAK1 play
critical roles in crossregulating NF-κB activation upon inflammatory stimuli (i.e. TNF-α, IL-
1β, LPS). However, the functional link between GSK-3 and TAK1 under these conditions remains to be investigated. Work from our group and others suggests that GSK-3 mediates
TNF-α-induced NF-κB activity at the transcriptional complex level (Hoeflich, Luo et al.
2000; Steinbrecher, Wilson et al. 2005). Additionally, we provide evidence that GSK-3 specifies NF-κB-dependent gene transcription at select promoters (Steinbrecher, Wilson et al.
2005). Interestingly, there is also evidence that p38 MAPK selectively regulates a subset of
NF-κB regulated genes upon inflammatory stimulation (Saccani, Pantano et al. 2002). This report suggests that p38 marks selected promoters for increased NF-κB recruitment by promoting histone H3 phosphorylation and phosphoacetylation (Saccani, Pantano et al.
2002). Numerous reports have demonstrated the requirement of TAK1 for inducible p38 activity upon inflammatory stimulation (Raman, Chen et al. 2007). Therefore, future studies
should investigate whether GSK-3 mediates TNF-α-induced TAK1 and subsequent p38 activity. Furthermore, these studies should utilize chromatin immunoprecipitation to
determine whether GSK-3 and TAK1 are required for histone H3 post-translational
modifications at select NF-κB-dependent promoters. The exact mechanism of how GSK-3
mediates inducible NF-κB activity has remained elusive over the past decade. These
proposed experiments will provide new insight into GSK-3-dependent NF-κB regulation and expand our understanding of the link between GSK-3 and TAK1.
(3) What role does nuclear GSK-3 and IKKα play in driving constitutive NF-κB
activity in pancreatic cancer cells? The previous chapters described a model by which
GSK-3 mediates constitutive NF-κB activity within the cytoplasm of pancreatic cancer cells.
112 However, evidence suggests GSK-3β (Ougolkov, Fernandez-Zapico et al. 2006; Ougolkov,
Bone et al. 2007) and IKKα (Anest, Hanson et al. 2003) can also function within the nucleus to mediate NF-κB transcriptional activity. Characterization of GSK-3β revealed a nuclear localization sequence (NLS) within the kinase domain between amino acids 85 – 123
(Meares and Jope 2007). Moreover, loss of GSK-3β kinase activity was associated with
decreased nuclear localization and proteasomal degradation of GSK-3β (Ougolkov,
Fernandez-Zapico et al. 2006; Meares and Jope 2007). A previous report demonstrated that
GSK-3β was aberrantly accumulated in the nucleus of human pancreatic tumor tissues and
pancreatic cancer cell lines (Ougolkov, Fernandez-Zapico et al. 2006). The role of nuclear
GSK-3α and GSK-3β in regulating constitutive NF-κB activity in pancreatic cancers remains
unclear. However, evidence in chronic lymphocytic leukemia cells suggest that GSK-3
inhibition can suppress NF-κB recruitment to target gene promoters through an epigenetic
silencing mechanism (Ougolkov, Bone et al. 2007). IKKα has also been reported to
translocate to the nucleus and promote chromatin remodeling at NF-κB target gene
promoters (Anest, Hanson et al. 2003). Importantly, the role of nuclear IKKα in regulating
constitutive NF-κB activity pancreatic cancer cells remains uncharacterized. Preliminary
data from our group indicates that IKKα, GSK-3α, and GSK-3β are present in the nucleus of human pancreatic cancer cells and tissue. Notably, we do not observe detectable levels of nuclear IKKβ. To determine whether nuclear IKKα and GSK-3 mediate constitutive NF-κB activity at the transcription complex level in pancreatic cancer cells, chromatin immunoprecipitation should be performed to detect recruitment of these kinases to known
NF-κB target promoters (i.e. IκBα, Bcl-XL, and cIAP2). Future studies should also ascertain
113 the requirement for nuclear IKKα and GSK-3 in mediating constitutive NF-κB activity.
Therefore, stable IKKα and GSK-3 null pancreatic cancer cells should be reconstituted with
mutant IKKα and GSK-3 that lack a functional NLS. These reconstitution experiments
would address whether these kinases are required for recruitment of NF-κB to target
promoter. Moreover, these studies would also determine the requirement of IKKα and GSK-
3 for transcriptionally active histone modifications (i.e. phosphorylation of histone H3 as
serine 10). Overall, the experiments described above may implicate a dual role for GSK-3
and IKKα in mediating constitutive NF-κB activity at the cytoplasmic and transcriptional
complex level in pancreatic cancer cells.
(4) Are the activities of GSK-3 and TAK1 required for pancreatic cancer
progression in vivo?
The previous chapters have demonstrated that GSK-3 and TAK1 play a key role in mediating
constitutive pro-survival NF-κB activity in pancreatic cancer cells. These data implicate
GSK-3 and TAK1 as potential therapeutic targets for pancreatic cancer. In order to pursue
this therapeutic strategy, further evidence is needed to establish the requirement of these
kinases within animal models. Furthermore, additional studies are needed to explore the
efficacy of GSK-3 and TAK1 inhibition on pancreatic cancer progression in vivo. GSK-3 inhibition (120 mg/ kg AR-A014418; every 12 hours for 2 days) was reported to suppress the tumor growth of an established CAPAN2 xenograft model (Ougolkov, Fernandez-Zapico et al. 2006). However, xenograft models do not mimic the unique tumor microenvironment necessary for pancreatic cancer progression. Therefore, alternative pancreatic cancer mouse models are needed to address the roles GSK-3 and TAK1 play during tumor progression.
Concomitant endogenous expression of KrasG12D and Trp53R172H in mouse pancreas
114 progenitor cells leads to accelerated pancreatic cancer development (Hingorani, Wang et al.
2005). Importantly, this animal model mimics human pancreatic cancer progression with
similar histological, clinical, and genetic phenotypes (Hingorani, Wang et al. 2005). Future
studies should utilize GSK-3 (AR-A014418) and TAK1 (5Z-7 oxozeanol) inhibitors to
determine the requirements of these kinases for pancreatic cancer progression and overall
survival. More importantly, the potential off-target effects these drugs may have on surrounding normal tissue should also be examined. Alternatively, studies should explore
whether targeted depletion of GSK-3α, GSK-3β, or TAK1 in pancreas progenitor cells could
attenuate pancreatic cancer progression within the KrasG12D, Trp53R172H animal model.
Ultimately, the experiments described above will provide the first steps necessary to
implement the development of GSK-3 and TAK1 targeted therapies for pancreatic cancer,
and their use in future clinical trials.
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