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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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