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Chapter 13
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DISCUSSION
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Chapter 13
Discussion
Amir Avan1, Thomas Würdinger
2, Gerrit Jan Schuurhuis
3, Godefridus
J. Peters1, Elisa Giovannetti
1
1. Department of Medical Oncology, VU University Medical Center, Amsterdam,
The Netherlands.
2. Neuro-oncology Research Group, Departments of Neurosurgery and Pediatric
Oncology/Hematology, VU University Medical Center, Amsterdam, The
Netherlands.
3. Department of Hematology, VU University Medical Center, Amsterdam, The
Netherlands.
Manuscript in preparation in conjugation with Chapter 1
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Discussion
An incidence rate nearly equal to its mortality rate demonstrates the aggressiveness and
lethal nature of pancreatic ductal adenocarcinoma (PDAC). Most patients present with
advanced disease (i.e., locally-advanced or metastatic) at diagnosis, and survival rate has
not improved in the last decade, with less than 5% of patients alive five years after
diagnosis. On one hand, such dismal outcome can be explained by the lack of biomarkers
for early screening/diagnosis, together with the aggressive biological behavior,
characterized by early metastatic spread and resistance to currently available chemotherapy
regimens. On the other hand, prognostic and predictive biomarkers of treatment response
are also missing, and most agents in clinical trials are selected on the basis of their activity
in preclinical models that do not recapitulate the molecular and histopathological hallmarks
of PDAC, thus creating a selection bias when choosing drugs for testing in the patients.
The research described in the current thesis was mainly focused on: (1) the elucidation of
molecular mechanisms underlying the aggressiveness and chemoresistance of PDAC, in
order to identify prognostic and predictive markers of treatment response; (2) the study of
the therapeutic potential of novel anticancer agents; (3) the establishment of innovative in
vitro and in vivo models of PDAC, starting from primary tumor cells, to test new (targeted)
treatment strategies (Figure 1).
Figure 1. Overview of the aims of the current thesis.
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Scope 1: Identification of prognostic or predictive markers of treatment response
Adjuvant or palliative chemotherapies slightly improve/prolong survival in the resected or
unresected PDAC patients. However, most patients do not achieve clinical response and
have a very short survival expectancy. Identification of novel predictive or prognostic
biomarkers of chemotherapy is essential for better clinical management. The aim of the first
part of the present thesis was to unravel predictive/prognostic biomarkers of chemotherapy
activity to select patients with the highest likelihood of responding, while minimizing
useless and toxic treatments, as described in chapters 2-6 and chapter 11.
Recently, the most relevant therapeutic improvement in metastatic PDAC has been obtained
from the combination of cytotoxic agents, such as 5-fluorouracil with leucovorin combined
with irinotecan and oxaliplatin, in the FOLFIRINOX regimen [1]. Moreover, Reni and
colleagues have reported the results of five studies [2-6], including a phase III trial,
demonstrating superiority over single-agent gemcitabine, of the four-drug regimens
cisplatin–epirubicin–5-fluorouracil–gemcitabine, cisplatin–docetaxel–capecitabine–
gemcitabine (PDXG) and cisplatin–epirubicin–capecitabine–gemcitabine (PEXG). Two
recent surveys mirroring the clinical practice in the first-line therapeutic management of
advanced PDAC suggested that four-drug combinations might yield a better outcome when
compared to other regimens [6,7]. However, combinations of several cytotoxic agents are
associated with increased hematologic or extra-hematologic side effects. Therefore, analysis
of accessible biomarkers, such as germ-line polymorphisms, could provide a useful tool in
the selection of the most appropriate chemotherapeutic regimen to be used in a patient-
adapted way.
1.1. XPD-Lys751Gln polymorphism as a prognostic factor in gemcitabine-cisplatin
polychemotherapy regimens in PDAC
In our previous pharmacogenetic study in 122 advanced PDAC patients treated with
gemcitabine–cisplatin-based polychemotherapy, the Xeroderma-Pigmentosum group-D
(XPD) polymorphism at codon-751 (XPD-Lys751Gln) emerged as the most significant
independent predictor for death- and progression risk, among 11 candidate functional
polymorphisms [24]. Consistent with these findings, we also showed a significant
association of XPD-Gln751Gln with shorter progression-free survival (PFS, P=0.02) and
overall survival (OS, P=0.04) in 93 advanced non-small cell lung cancer patients treated
with second-line carboplatin plus pemetrexed [8]. However, a recent meta-analysis of 12
studies demonstrated inconclusive data about this polymorphism in NSCLC [9]. These
discrepancies could suggest that pharmacogenetic associations are not always reproducible
in small size studies, with different clinical settings, tumor types, stage and treatment.
Larger multicenter studies are essential to investigate the role of emerging biomarkers
before planning of prospective trials.
Therefore in the chapter 2 of this thesis, we further investigated the prognostic role of
XPD-Lys751Gln in 337 patients (247 treated with gemcitabine–platinum regimens and 90
treated with gemcitabine) with locally advanced or metastatic PDAC. Moreover, to test our
hypothesis that XPD-Gln751Gln genotype was associated with a more efficient DNA repair
after cisplatin exposure, we determined DNA damage capacity in lymphocytes harboring
the different XPD-Lys751Gln genotypes.
In this study, we identified XPD-Lys751Gln polymorphism as a significant independent
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predictor for death and progression-risk in PDAC patients treated with four-drug
polychemotherapy regimens. In particular, patients carrying the XPD-Lys751Lys or
Lys751Gln genotypes had a significantly longer median OS (log-rank-P<0.01). Similarly,
the median PFS of patients harboring the XPD-Gln751Gln genotype was significantly
shorter than the median PFS of XPD-Lys751Lys or XPD-Lys751Gln patients. Moreover,
The XPD-Gln751Gln genotype was markedly associated with increased risk of death
(HR=2.1) as well as with increased risk of progression (HR=1.9) at multivariate analysis. In
addition, the analysis of DNA damage in lymphocytes supported the association of XPD-
Gln751Gln with greater resistance to cisplatin-induced damage. These results might be
explained by the central role of XPD in DNA-repair and platinum activity, and may have
important clinical applications. Indeed, the analysis of the polymorphism by a simple blood
test offers an innovative tool for optimizing palliative chemotherapy in patients with
advanced PDAC.
1.2. EZH2 is a prognostic factor for locally-advanced and metastatic PDAC
Several genetic alterations have been associated with the aggressive behavior and
chemoresistance of PDAC [77], while epigenetic factors recently emerged for their roles in
tumor progression. In this context, Enhancer of Zeste Homolog 2 (EZH2) is becoming
increasingly acknowledged as a prognostic biomarker in radically resected PDAC patients
[11]. Therefore in the chapter 3, we describe the prognostic value of EZH2 in the PDAC
patients with locally advanced or metastatic disease. Moreover, since recent studies
suggested a role for candidate polymorphisms of EZH2 in lung cancer risk and colorectal
cancer prognosis [12,13], we investigated the correlation of candidate polymorphisms and
EZH2 expression with outcome. EZH2 mRNA and protein levels were evaluated in two
cohorts of 32 laser-microdissected specimens and 25 samples collected in a Tissue
Microarray (TMA), while polymorphisms analyses were performed in 340 patients (247
treated with four-drug regimens, as reported in the previous chapter 2, and 93 treated with
gemcitabine).
Patients were divided into two subgroups according to the median EZH2 mRNA expression
and evaluated for clinical outcome after gemcitabine chemotherapy. The high EZH2
expression group had a significantly poorer prognosis. Immunohistochemistry showed a
variable protein expression in the patient samples, related to the mRNA expression. Indeed,
the tissues characterized by high EZH2 expression, showed a strong and diffuse staining,
while the tissues with low EZH2 expression had only few scattered positive cells with a
weak nuclear staining. EZH2 protein expression was also related to outcome, and similar
results were observed in the TMA samples. EZH2 expression was lower in grade-I/II
(N=13) than grade-III (N=19), while no difference was observed according to other
clinicopathological parameters. In addition, the rs6950683 C/C genotype was associated
with a markedly higher EZH2 expression, and patients harboring this genotype had a trend
towards a significantly shorter OS. However, no significant differences were observed in
OS for EZH2 polymorphisms in two larger cohorts of patients, treated with gemcitabine-
alone and with polychemotherapeutic regimens from a multicentric series. In conclusion,
EZH2 expression emerged as a prognostic factor for locally advanced or metastatic PDAC,
but candidate polymorphisms could not predict the outcome. Other factors involved in the
EZH2-oncogenic pathways and detectable in accessible samples sources, such as candidate
miRNA (ie. miR-101) enriched tumor-derived exosomes in peripheral blood [14], should be
investigated in order to improve the clinical management of advanced PDAC patients.
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1.3. Do observational studies provide a strong rationale for future trials to validate the
best markers for personalized treatment of PDAC patients?
Recently, Fisher and colleagues showed that high tumor expression of ribonucleotide
reductase subunit-M2 (RRM2) and excision repair cross-complementing group-1 (ERCC1)
correlated with reduced survival, as determined by immunohistochemistry [15]. In chapter
4, we discuss these findings in comparison with our previous results. In particular, in our
previous studies on mRNA expression of 7 genes involved in gemcitabine activity in laser-
microdissected PDACs, we did not observe differences in RRM2 expression using a
specific quantitative-RT-PCR technique, while patients with higher levels of human
equilibrative nucleoside transporter-1 (hENT1) had significantly longer OS [16]. In
agreement with these data, other studies showed that patients with high hENT1 expression
benefit from gemcitabine-based adjuvant chemotherapy [17-19]. However, a recent study
indicates a lack of association with hENT1 expression in the prospective multicenter
NCT01124786 trial. The discrepancies observed might be due to the use of different
methods, treatments heterogeneity, and relatively small sample size. Standardized
techniques of sample collection/processing, larger and uniformly treated populations and
integration with functional data, are crucial to validate the best markers for personalized
treatment of PDAC patients.
1.4. MicroRNA-211 as a prognostic factor in resected PDAC
The role of RRM2 expression was further investigated in our following studies on
microRNAs (miRNAs), since it is one of the targets of miR-211, as described in chapters 5
and 6. Several studies have evaluated the complex genetic networks and transcriptomics
alterations underlying the development and progression of PDAC [20,21]. The recent
discovery of miRNAs has provided additional insights potentially explaining the gap that
exists between tumor genotype and phenotype. MiRNAs play essential roles in the control
of proliferation, differentiation and apoptosis, while their aberrant expression in many
tumors, indicated that they might function as oncogenes or tumor suppressor genes [22,23],
suggesting their use for diagnostic and therapeutic purposes. Moreover, since miRNAs are
stable and detectable in human blood they could be useful as diagnostic or prognostic
markers. Chapter 5 of this thesis reviews the role of miR-211 in PDAC as well as in other
human diseases. The role of this miRNA in PDAC emerged from a comprehensive miRNA
expression profiling of more than 1200 human miRNA performed to distinguish resected
PDAC patients with short OS (≤12 months) from long term survivors (>30 months), as
described in chapter 6. This extensive miRNA microarray analysis resulted in a list of 170
miRNAs that show significant differences in expression between the two groups. RELIEF
algorithm and highly stringent statistics identified the top 4 discriminating miRNAs (miR-
211, miR-1207-3p, miR-326 and miR-4321) between patients with short- vs. long-OS. We
investigated the expression of these miRNAs by RT-PCR in an additional cohort of
patients. Patients with low miR-211 expression had a significantly shorter median OS (14.8
months, 95%CI, 13.1–16.5 months) compared to patients with high miR-211 expression
(median OS, 25.7 months, 95%CI, 16.2–35.6 months, HR= 3.0, 95%CI, 2.1–8.9, P=0.001).
Similar results were found for the disease free survival (DFS) of patients with low miR-211
expression, who had a median DFS of 16.7, compared to 9.3 months in patients with high
miR-211 expression (P=0.004). The Cox proportional hazards regression model used for
the multivariate analysis illustrated the prognostic significance of miR-211 expression and
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grading. Further in vitro studies were performed on the expression and role of miR-211 in
PDAC cells. MiR-211 was expressed in all the PDAC cell lines and primary tumor cell
cultures, and its modulation was related to gemcitabine cytotoxicity. Induction of the miR-
211 expression in the cells increased the sensitivity to gemcitabine and reduced the
expression of its target RRM2.
In conclusion, our studies identified some novel prognostic and predictive markers,
including polymorphisms and miRNA expression. However, future studies should validate
these candidate markers in larger cohorts, ideally in the prospective and multicenter setting.
Randomized studies with a control arm of patients treated with other regimens and the
comparison of the survival stratified by miRNA expression would be the only way to
establish their predictive role. Moreover, these studies should also implicate the potential
use of our candidate biomarkers in the neoadjuvant setting, which might provide an
alternative for patients with aggressive disease, who could be given chemotherapy before
surgery to kill any micrometastases.
Scope 2: Therapeutic potential of novel anticancer agents in treatment of PDAC
Recent advances in genome sequencing have identified a complex picture of genetic
aberrations in the initiation and progression of PDAC [20,21]. These genetic alterations
influence the activity of core signaling pathways (e.g., Akt/PI3K and Met/HGF pathways),
which are altered in the majority of analyzed PDACs, representing targets for novel
therapeutic strategies. Of note, also some of the markers that were evaluated in our previous
studies, such as EZH2 (chapter 2), might be targeted by new drugs, as described in
chapter 7. Therefore, the main aim of the second part of this thesis was to evaluate new
agents for treatment of PDAC, as described in chapters 7-10 and summarized below.
2.1. Inhibition of EZH2 reduces aggressiveness of PDAC and enhances sensitivity to
gemcitabine
Recently PDAC emerged as a Cancer Stem Cell (CSC) – driven disease. Pancreatic CSCs
are highly tumorigenic and have the abilities to self-renew and produce differentiated
progeny. CSCs have also been associated with chemoresistance to gemcitabine [25,27].
Against this background, studies on key determinants in CSCs can provide both biomarkers
of the aggressiveness of PDAC and novel targets to overcome chemoresistance. EZH2 is a
histone methyltransferase essential for self-renewal of CSCs [28]. In chapter 7, we
evaluated the EZH2 expression in PDAC tissues and cells, and investigated the cell growth
inhibitory effect of the EZH2 inhibitor DZNeP in combination with gemcitabine in
monolayer cell cultures and cells growing as spheroids in serum-free-CSC-medium. EZH2
was expressed in all our PDAC cells, including 7 primary tumor cell cultures, while the
expression was significantly lower in both fibroblasts and normal pancreatic ductal cells
HPNE. DZNeP significantly reduced EZH2 and H3-K27 expression and we also showed
that its combination with gemcitabine was synergistic. This synergistic interaction against
cell proliferation was associated with a significant increase in apoptosis induction.
However, our findings showed that this synergistic interaction is also mediated by other
mechanisms, which reduced the aggressiveness of PDAC and enhanced sensitivity to
gemcitabine. Consistent with previous studies showing that inhibition of EZH2 by DZNeP,
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attenuated glioblastoma and mesothelioma cell migration/invasion [29], our data showed a
marked reduction of cell migration. Keeping with previous findings on inverse relationship
between EZH2 and E-cadherin expression [10], our data showed that DZNeP-induced
EZH2 inhibition resulted in an increase in both mRNA and protein expression of E-
cadherin. Moreover, DZNeP significantly reduced the growth of PDAC spheroids in serum-
free-stem-cell medium and effectively depleted the most aggressive subpopulation of PDAC
cells, as suggested by the significant reduction of CSC-marker CD133 expression.
Moreover, we analyzed the expression of key nucleoside transporters (hENT1 and hCNT1),
showing that DZNeP significantly increased the expression of these transporters. These
findings can be explained at least in part by the reduction of endogenous deoxynucleotides,
which might determine the up-regulation of both hENT1 and hCNT1, potentially
facilitating gemcitabine cytotoxicity.
In conclusion, inhibitors of EZH2, such as DZNeP, seem promising anticancer agents, by
attacking key mechanisms involved in the proliferation, cell cycle control, apoptosis as well
as migration properties of PDAC cells. All these molecular mechanisms underlying the
synergism of DZNeP/gemcitabine combination, support further studies on this novel
therapeutic approach as well as on novel anti-EZH2 compounds, with a better
pharmacokinetic profile than DZNeP, to improve the efficacy of the actual treatment of
PDAC.
2.2. Inhibition of Akt/PI3K signaling pathway increases the chemosensitivity of PDAC
cells to gemcitabine
The Akt/PI3K pathway is one of the core signaling pathways affected in PDAC [20]. Akt is
overexpressed in more than 40% of PDAC patients [30,31], and has been shown to be
associated with PDAC poor prognosis and chemoresistance [32]. The Akt/PI3K pathway
regulates tumor-associated cell processes such as cell growth, cell cycle progression,
survival, migration, epithelial–mesenchymal transition (EMT) and angiogenesis [33]. Fahy
and colleagues showed that inhibition of the PI3K/Akt pathway sensitizes PDAC cells to
the apoptotic effect of PI3K or Akt inhibitor both in vitro and in vivo [33]. Therefore, we
investigated the therapeutic potential of the novel Akt inhibitor perifosine in combination
with gemcitabine in PDAC cells in chapter 8.
Perifosine is a synthetic alkylphosphocholine that inhibits Akt activation by targeting the
pleckstrin homology domain of Akt [34]. Anti-tumor activity of this drug has been observed
in a variety of cancers in vitro and in vivo [35] and its clinical efficacy was evaluated in
phase II/III clinical trials in patients with advanced solid tumors, including breast and
prostate cancers [36,37]. Unfortunately, a phase II study in locally advanced, unresectable,
or metastatic PDAC failed, as a result of unacceptable adverse events [38]. Therefore we
evaluated the therapeutic potential of this inhibitor in several representative PDAC cells and
primary PDAC cells, in order to identify biological factors that can be used to tailor this
treatment. We observed that perifosine inhibited cell growth and interact synergistically
with gemcitabine in PDAC cells with high expression of Akt, while an antagonist
interaction was observed in cells with low Akt expression. The synergistic effect was
associated with reduction of the expression of RRM1 and RRM2, potentially facilitating
gemcitabine cytotoxicity. Furthermore, this synergistic effect was associated with inhibition
of growth of PDAC spheroids. In line with the inverse relationship between Akt and E-
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cadherin expression [39], our results showed that perifosine increased the expression of E-
cadherin in the PDAC cells.
Since the Akt pathway plays an important role in cell survival process, its blockage can
result in activation of programmed cell death [40]. Therefore, we also evaluated the effect
of perifosine on cell cycle perturbation and apoptosis induction, showing significant
(P<0.05) enhanced apoptosis in PDAC cells with high Akt expression. This was associated
with activation of a number of pro-apoptotic markers, including caspase -3/-6/-9, BAD and
PARP, and inhibition of NF-kB and Bcl-2 expression.
In aggregate, our data provide novel insights in the antitumor activity of perifosine in
PDAC, supporting the analysis of the expression of Akt and other biomarkers for the
rational development of this therapeutic approach.
2.3. Antitumor activity of novel c-Met/ALK inhibitor crizotinib in PDAC
The receptor tyrosine kinase Mesenchymal-Epithelial Transition factor (c-Met) plays
essential roles in embryonic and adult life, including embryonic development, tissue
homeostasis and morphogenesis [41-43]. Conversely, abnormal stimulation of this pathway
contributes to cellular transformation, epithelial-to-mesenchymal transition, tumor invasion,
progression and metastasis [44,45]. The c-Met/HGF signaling pathway is aberrantly
activated or overexpressed in a variety of solid tumors, including PDAC [43,46-51]. Of
note, c-Met is expressed in the developing pancreatic bud of the embryo and marks
candidate stem/progenitor cells in the embryonic and adult pancreas, but it is expressed at
very low levels in normal adult differentiated pancreatic cells [52]. However, the MET gene
is amplified or overexpressed in progenitor ductal cells [53] and emerged as a stem cell
marker in pancreatic tissues [54] as well as a marker of pancreatic CSC [51]. Moreover,
interactions between cancer cells and fibroblasts through c-Met increased PDAC
invasiveness [55,56], and factors affecting the cancer/stroma interaction play a key role in
PDAC progression and aggressive behavior [57]. Other studies demonstrated that PDAC
cells overexpressing c-Met are chemoresistant to gemcitabine [58,59].
Therefore, in chapter 9 of the thesis, we describe the critical role of the HGF/c-Met
signalling pathway in upper gastrointestinal cancers, as well as the preclinical and clinical
investigations on c-Met inhibitors in solid tumors, with particular emphasis on recent
findings with small-molecule inhibitors in PDAC. As summarized in this review, a variety
of different strategies to inhibit this signaling pathway have been investigated ([1] inhibition
of HGF, and [2] inhibition of Met with Met antibodies or [3] tyrosine kinase (TK)
inhibitors), and a large number of new molecules entered preclinical and clinical
investigations. Crizotinib has recently been approved for ALK-rearranged non-small cell
lung cancer, while the clinical efficacy of tivantinib needs to be ultimately validated in
ongoing phase III randomized trials. On the other hand, several important questions remain
to be unanswered on the molecular mechanisms underlying the antitumor effects of these
drugs, as well as on their possible role in combination treatments of different tumor types,
including PDAC.
Using the knowledge gained from the chapter 9, the therapeutic potential of the c-
Met/ALK TK inhibitor crizotinib was tested alone and in combination with gemcitabine in
PDAC cells including the Capan-1-gemcitabine-resistant cells (Capan-1-R), as described in
chapter 10.
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Recently, pancreatic CSCs have been shown to be associated with the aggressiveness of
PDAC, metastatic behavior and intrinsic resistance to chemotherapy [51,60]. Li and
colleagues identified a subpopulation of highly tumorigenic cancer cells expressing the cell
surface markers CD44, CD24, and CD326 [61]. Consistent with these findings, our results
illustrated that the expression of CD44, CD24 and CD326 were increased at the mRNA and
protein levels in Capan-1-R compared to Capan-1 cells. Moreover, these cells have higher
expression of c-Met and phospho-c-Met, suggesting that the c-Met signaling pathway could
be a valuable target to overcome chemoresistance. Therefore, we evaluated the
pharmacological interaction of crizotinib with gemcitabine, showing that this combination
was synergistic. In addition, crizotinib down-regulated the expression of
CD44+/CD133+/CD326+, as well as that of c-Met.
We also evaluated the expression of EMT markers showing that levels of E-cadherin were
not affected, while vimentin expression was increased in Capan-1-gemcitabine-resistant
cells compared to Capan-1 cells. These results are in agreement with a previous study,
showing that gemcitabine-resistant cells were more invasive and migratory and had
increased vimentin expression [62]. Notably, crizotinib significantly reduced vimentin
expression, resulting in an impaired migration, which might be attributed, at least in part, to
a reversal of their EMT phenotype.
Finally, we investigated the expression of the main determinants of gemcitabine activity in
Capan-1-R and Capan-1 cells, as well as the effect of crizotinib on the modulation of these
genes. These results showed that the mRNA expression of hCNT1, deoxycytidine kinase
and RRM1 and RRM2 were significantly reduced in Capan-1-R cells compared to Capan-1.
Conversely, the mRNA expression and enzyme activity of cytidine deaminase (CDA) in
Capan-1-R cells were significantly higher than in Capan-1 cells. This can potentially
explain the significantly lower levels of gemcitabine metabolites in Capan-1-R compared to
Capan-1. Moreover, crizotinib markedly decreased the CDA expression and increased
hCNT1 expression, potentially reducing gemcitabine catabolism, while increasing
gemcitabine uptake.
In conclusion, our findings provide novel insights into the antitumor activity of crizotinib in
PDAC cells, unraveling its ability to specifically target CSC-like-subpopulations, interfere
with cell-proliferation, induce apoptosis, reduce migration and synergistically interact with
gemcitabine, supporting further studies on this novel therapeutic approach for PDAC.
Therefore, we further explored the therapeutic potential of this therapeutic regimen in
orthotopic PDAC mouse models derived from primary PDAC cells, as described in the
Chapter 11.
Scope 3: Development of new patient-derived orthotopic mouse PDAC models:
Towards new treatment strategies
As discussed above, some of the unfavorable clinical factors, which may have at least in
part, hampered clinical progress in PDAC include: (1) selection of most anticancer drugs in
clinical trials on the basis of their activity in preclinical models that do not recapitulate the
complex molecular and histopathological hallmarks of PDAC, and (2) lack of appropriate
drugs that combine synergistically with gemcitabine.
In the context of preclinical models, the most commonly used PDAC models include 1)
established cell line xenografts cultured as monolayers or grown as their xenografts, and 2)
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genetically engineered mouse models (GEMMs). Unfortunately, culturing of the PDAC
cells in plastic-flask for long term could lead in irreversible loss of important genetic and
biological properties, including complex genomic aberrations affecting critical signaling
pathways [20], maintenance of a distinct stem cell population [51;63], and ability of the
PDAC cells to metastasize [64]. GEMM provides an important tool to unravel the function
of specific genes in tumor initiation and progression [65]. These models also have the
advantage to maintain an intact immune system and present tumors histologically similar to
human PDACs, including a dense desmoplastic stroma reaction [57]. However, several
promising targets identified through GEMMs do not have human counterparts, while other
important genes in human PDAC are not expressed in mice [66]. In addition, these models
depend on a few critical genetic lesions, e.g., mutations in K-Ras, P53, and CDKN2A/P16,
which may not completely recapitulate the genetic diversity of human PDAC.
Voskoglou-Nomikos and colleagues suggested that early passages of primary PDAC cells
may better mimic the genetic characteristics of the disease and might be better predictors of
drug activity [67]. Several subcutaneous mouse models have been developed by implanting
pieces of human PDACs into mice [68,69]. However, it has been shown that
subcutaneously implanted tumors may not optimally recapitulate many of the essential
features of tumor growth in patients, such as the ability to metastasize [70].
Therefore, we aimed to develop orthotopic xenograft models employing low passage
primary PDAC cells in order to preserve the genetic background and heterogeneity of
human PDAC, as well as to maintain the macro- and micro-environmental interactions, as
described in chapter 11. The cells were genetically engineered to express Firefly- and
Gaussia-luciferases to provide a reliable indicator for localizing and quantifying orthotopic
pancreatic tumors and metastases [71].
3.1. Crizotinib inhibits metabolic inactivation of gemcitabine in c-Met-driven pancreatic
carcinoma
We have established 4 primary PDAC cell cultures (PDAC-1/-2/-3/-4) from surgically
resected tumor masses using sterile non-necrotic tumor samples (40% efficiency). These
cells were successfully co-transduced with two lentiviral vectors, expressing Fluc-mCherry
(FM) and Gluc-CFP (GC) and then injected orthotopically into the pancreas of three
athymic mice. All four primary transduced PDAC cell cultures engrafted and developed
tumors in all the mice injected (100% take rate) and expanded over time, as determined by
the increase in Fluc and Gluc signal intensities. Remarkably, survival trend observed in the
mice followed a same trend in the PDAC patients, although the number of models does not
warrant a statistically supported survival correlation. Moreover, MR-scans confirmed the
localization of tumor cells in the pancreas, as well as retroperitoneal invasion, while high-
frequency-ultrasound enabled for the measurement of tumor volumes and revealed
hypovascular pancreatic tumors.
All these models showed the main histopathological features of human PDAC, compared to
surgical material resected from the human originator tumor, including tumor infiltration,
stroma and PDAC-associated desmoplastic reaction, ductal characteristics, adenocarcinoma
differentiation, and inflammation. In addition, the xenografts stained positively with human
specific antibodies, which are routinely used in PDAC immunohistochemistry. Moreover,
we evaluated the hypovascular and hypoxic areas by analysis of CD31 and Carbonic
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Anhydrase IX (CAIX), respectively [72,73]. These results showed that the blood vessels
were organized in the stroma surrounding the tumor nests and did not invade into the tumor.
Similarly, most tumors showed high expression of the hypoxia marker CAIX. Importantly,
most of the orthotopic tumors metastasized to other organs and followed the typical routes
of invasion to lymph nodes, liver and lung, as observed in patients.
After finding a similar phenotype, we investigated whether the genetic signature of the
human originator tumors was preserved. The orthotopic PDAC models showed similar
genomic abnormalities as their human originator tumors, and genotypic heterogeneity was
reflected by an average of more than 50 different aberrations, as detected by high-resolution
array comparative genomic hybridization (aCGH) and mutation studies. These models can
therefore be used as reliable tools for understanding the role of complex PDAC genetic
characteristics and testing new targeted drugs.
Interestingly, in one of our PDAC specimens, PDAC-3, we observed a high copy number
gain of the c-MET gene. These results were validated by copy number analysis as well as by
gene and protein expressions analysis. Then we tested the activity of three novel c-Met
inhibitors, tivatinib, crizotinib and DN-30. All these compounds were more effective in the
PDAC-3 cells compared to other PDAC cells, and crizotinib emerged as the most active
inhibitor of cell growth. The cells were also treated with the combination of crizotinib and
gemcitabine, median drug-effect analysis showed strong synergism in these cells, in parallel
with increase in the accumulation of gemcitabine-nucleotides. Since previous studies
suggested that gemcitabine is inactivated by CDA [74,75], we investigated CDA activity in
the cells upon treatment with crizotinib, showing a significant reduction in CDA activity.
This reduction was explained by the degradation mediated by crizotinib-induced ROS, as
determined by analysis of reactive oxygen species (ROS) activity. This might at least in part
explain the synergistic effect.
Furthermore, crizotinib significantly reduced tumor growth in PDAC-3-FM-GC mice,
which survived longer than untreated controls. Moreover, liquid chromatography-tandem
mass spectrometry analyses demonstrated significantly higher concentrations of
gemcitabine in the tumors and blood samples from mice treated with the gemcitabine-
crizotinib combination compared to gemcitabine alone.
In conclusion, with the establishment and extensive genetic and histopathological
characterization of our double bioluminescent patient-derived orthotopic mouse PDAC
models, we provided novel preclinical models to explore therapeutic strategies and
mechanistic insights that can ultimately be applied to the future clinical practice for the
individualized treatment of PDAC patients. Here we used our models to identify crizotinib
and gemcitabine as a promising drug combination, acting synergistically via simultaneous
targeting of key intratumoral genetic features and increase of drug delivery by CDA
modulation, and warranting further investigation for the treatment of PDAC.
3.2. CYB5A as a novel prognostic factor for PDAC, exerting its tumor-suppressor
function via autophagy-induction and TRAF6 modulation
Our models were also used in a study aiming at the identification of novel prognostic
biomarkers. A recent study showed the correlation of genomic imbalances and clinical
outcome using aCGH in 44 radically resected patients [76] (Lee et al., 2012), demonstrating
a significant association between shorter survival and loss of the small cytoband 18q22.3.
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Therefore, in the chapter 12, we further evaluated whether the mRNAs and/or proteins
encoded by the genes in the 18q22.3 cytoband were associated with the outcome of PDAC
patients in two homogeneous cohorts of radically resected patients. Moreover, we explored
the role of CYB5A in primary tumor growth and metastatic spread using an orthotopic
PDAC mouse model.
The genes located in the 18q22.3 cytoband (FBXO15, C18orf55, CYB5A, CPGL, and
CPGL-B transcripts) were detectable in most samples, however median expression values
of CYB5A and C18orf55 were significantly lower in deleted versus non-deleted samples.
Moreover, patients with tumors expressing low levels of the CYB5A gene had a shorter OS
than the high expression group (HR=2.3, P=0.01). Similar results were observed for the
DFS curves. The prognostic role of CYB5A was then validated by IHC analysis of
specimens from an independent cohort of 100 radically resected PDAC patients, collected
in a tissue-microarray. The multivariate analyses demonstrated that CYB5A expression was
an independent prognostic factor, and lower expression levels of CYB5A were correlated
with an increased risk of death (HR=2.0, P=0.02), and relapse (HR=1.8, P=0.03).
Then we investigated the role of CYB5A in PDAC biology; we initially assessed the
CYB5A expression in 11 PDAC cell lines, 5 primary cell cultures and in the immortalized
normal ductal epithelial HPNE cells, showing lower expression levels of CYB5A in all of
the malignant cells, compared to the non-tumorigenic control. Su86.86 and PDAC-2 cells
were selected for further studies, as they both carried the 18q22.3 cytoband loss. We
successfully established CYB5A overexpressing stable subclones and empty vectors
(named CYB5A+, and CTR respectively). We demonstrated that CYB5A retrovirus-
mediated up-regulation reduced proliferative and invasive capacity while increasing
autophagy induction, as determined by electron microscopy. Furthermore, in order to shed
light on the molecular events driving autophagy, we performed a PCR array on 84 key
autophagic genes, revealing a significant up-regulation of BAX, accompanied by down-
regulation of BCL-2 and MAPK14, while a kinase array revealed the down-regulation of
phospho-EGFR and phospho-MAPK14. Network analyses suggested the interaction of
CYB5A with TRAF6, a molecular bridge for many diverse signals, both upstream and
downstream, including Akt and MAP kinases, as well as with several genes involved in cell
death. Both transduced PDAC-2- and Su86.86 cells had a marked reduction of TRAF6
levels, which was restored in a rescue experiment with the transfection of CYB5A siRNA.
Finally, we tested the tumor-suppressor function of CYB5A in our orthotopic mouse
models derived from the CYB5A+ retrovirus-transduced cells, which had increased
survival, as well as reduced primary tumor growth and metastatic spread. Moreover, the
tumors in the CYB5A+ group had a weak expression of TRAF6 with respect to the
moderate/strong staining in the CTR group.
In conclusion, we identified CYB5A as a novel inhibitor of anti-autophagy oncogenic
phenotypes, raising the possibility at restoring CYB5A activity, via gene therapy, or
targeted therapy inhibiting its potential downstream TRAF6. This might constitute a novel
approach that interferes with multiple signalling components of EGFR, TGF-β, Akt and
Src, favouring cancer cell death while preventing potentially deleterious cross-talk between
these pathways in specific subgroups of PDAC patients.
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Conclusions and Future Directions
The studies described in this thesis show that several genetic and epigenetic alterations are
the main driving forces of PDAC progression and metastasis. Understanding these
alterations is the first step on the road to improve the current outcome. The signaling
pathway affected by these alterations should be the target for tailored treatment of these
patients. In particular, here we demonstrate that c-Met/HGF and Akt/PI3K pathways
constitute potential important targets. Targeting the stromal compartment of the tumor is
another new promising strategy that may improve the poor prognosis of PDAC. Further
investigations are needed to identify and select the optimal patient populations that will
benefit from specific treatments. However we reckon that our investigations could provide
innovative tools for further progressing in treatment of this disease.
It is obvious that there is still a long way to go for the finding a cure for this devastating
disease. For future years, it is hoped that insights from preclinical and translational studies
will result in improvement of treatment and survival and hopefully the results of the
research described in the current thesis contribute to that.
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