The role of hypoxia in colon cancer cell

resistance to cytotoxic antitumour agents and

modulation of Hypoxia-inducible factor-1 as

a strategy to circumvent chemoresistance.

Brecht QUINTENS

Master’s thesis submitted

to obtain the degree of

Master in the Biomedical Sciences

Promoter: Prof. Dr. J. Gettemans

Department of Medical Protein Research,

Ghent University

Co-Promoter: Prof. Dr. E. Monti

Department of Structural and Functional Biology,

University of Insubria

Erasmus Programme Academic year: 2008-2009

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2

The role of hypoxia in colon cancer cell

resistance to cytotoxic antitumour agents and

modulation of Hypoxia-inducible factor-1 as

a strategy to circumvent chemoresistance.

Brecht QUINTENS

Master’s thesis submitted

to obtain the degree of

Master in the Biomedical Sciences

Promoter: Prof. Dr. J. Gettemans

Department of Medical Protein Research,

Ghent University

Co-Promoter: Prof. Dr. E. Monti

Department of Structural and Functional Biology,

University of Insubria

Erasmus Programme Academic year: 2008-2009

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4

Preface

This thesis is a part of the Erasmus exchange project among the universities of Insubria and

Ghent. I have performed a 7 month internship at the laboratory of Prof. Dr. E. Monti located

in Busto Arsizio, which is specialized in anti-cancer pharmacology.

First of all I thank my co-promoter Prof. Dr. E. Monti for the excellent guidance throughout

the project as well as Prof. Dr. M. Gariboldi, Dr. R. Ravizza and last but not least Dr. R.

Molteni, each of them possessing great theoretical and practical skills, and, a lot of patience

and devotion to let me acquire the variety of laboratory - and cell techniques. Apparently this

also applies for the other lab-members who even inspired me to learn the Italian language.

I would like to thank Prof. Dr. G. Perletti for his concern as Erasmus responsible, in fact, I

consider him as my ‘Erasmus-dad’; hotel-labo shuttle services, sport material, nothing is too

much for him. His Belgian counterpart, Prof. Dr. J. Gettemans who is Erasmus responsible for

the Biomedical Sciences at UGhent and also my promoter, would I like to thank thoroughly

because of the many arranged essential things, including the traineeship in the first master.

Thanks both of you!

I also want to thank my parents - not for doing my laundry and catering services the last 8

months - , but for giving me this and other opportunities.

It’s not possible to end without mentioning Serge Hoefeijzers, my ‘Erasmus-buddy’. We have

gone through the same things and helped one another where useful, or where compulsory

(laundry for instance) and we strengthened each other as individuals, hence we were able to

finish this academic year.

As a biomedical scientist I am supposed to end with a conclusion:

Personally I experienced whole this Erasmus-programme as enriching, fantastic and intense in

both a cultural and scientific point of view!

Brecht Quintens, Milan, 20th

May 2009.

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Index

Abstract...................................................................................................................................1

Introduction...........................................................................................................................2

1. Colon cancer

1.1. Epidemiology and risk factors

1.2. Genetic basis and pathogenesis....................................................................3

1.3. Therapeutic strategies...................................................................................5

1.3.1. Oxaliplatin

1.3.2. 5-Fluorouracil..............................................................................8

1.3.3. Cetuximab & Bevacizumab.......................................................11

1.3.4. Combination strategies

2. Colon cancer & Hypoxia.......................................................................................12 2.1. Hypoxia

2.2. Hif-1 structure and regulation....................................................................15 2.2.1. Oxygen dependent HIF-1 regulation

2.2.2. Oxygen independent HIF-1 regulation......................................17 2.3. HIF-1 and drug resistance..........................................................................18

3. Modulation of HIF-1.............................................................................................19 3.1. PMX290.....................................................................................................20

3.2. Antisense oligonucleotides

4. Aims........................................................................................................................21

Materials & Methods........................................................................................................23

Results....................................................................................................................................29

1. Effect of hypoxia on HIF-1α expression

2. Effect of hypoxia on HIF-1α activity

3. Effect of hypoxia on cellular response to 5FU and OxPt........................................30

4. Effect of modulation of HIF-1α on the response of colon

cancer cells to 5FU and OxPt..................................................................................34 5. Effect of EZN2969 treatment on HIF-1α expression, HIF-1

activity and apoptotic response to 5FU in HCT116cells.........................................38 6. Effects of the expression of a degradation-resistant variant

of HIF-1α on the response of HCT116 cells to 5FU...............................................39

Discussion..............................................................................................................................41

1. 5-Fluorouracil

2. Oxaliplatin...............................................................................................................44

3. General Conclusion.................................................................................................46

References.............................................................................................................................47

1

Abstract

Tumour hypoxia represents a major obstacle to the success of chemotherapy. Hypoxia-

inducible factor 1 (HIF-1) is the pivotal agent of cellular response to low oxygen levels,

which is apparent in colon cancers, and these facts led to the concept that inhibiting HIF-1

activity may sensitise hypoxic colon cancer cells to cytotoxic drugs.

In this study, we investigate the effects of HIF-1 modulation on the response of two human

colon adenocarcinoma cell lines, HCT116 and HT29, which differ in p53 status, to either 5-

fluorouracil (5FU) or oxaliplatin (OxPt), both currently used in the treatment of colon cancer.

Increasing HIF-1 activity, either by exposing the two cell lines to hypoxia or by forced

expression of a degradation-resistant form of HIF-1α in HCT116 cells, results in poor cell

response to 5FU; conversely, knockdown of HIF-1α by antisense oligonucleotides targeting

the HIF-1α mRNA prevents hypoxia-induced resistance to 5FU. PMX290, a thioredoxin-1

inhibitor, significantly inhibits HIF-1 activity and concomitantly sensitises both HCT116 and

HT29 hypoxic cells to the cytotoxic effect of 5FU and OxPt. Moreover, these results were

confirmed in HCT116 cells grown as three-dimensional spheroids, a model that more closely

reproduces the hypoxic environment of solid tumours. Based on these observations,

downregulation of HIF-1 activity is a potential approach to the circumvention of

chemoresistance in the clinical management of colon cancer.

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Introduction

1. Colon cancer

1.1. Epidemiology and risk factors

Colon cancer is the third most common cancer in the world in terms of prevalence and there is

a worldwide mortality/incidence ratio of 52 % [1, 2]. In Flanders (Belgium) the 5-year

survival is 57 %, but this ranges from 65 % in North-America to 30 % in India, indicating a

substantial variation in therapeutic strategies and/or options [3, 4]. So there is a general

relatively good prognosis, nevertheless more than half a million people die each year of

colorectal cancer due to the high incidence. Unlike most locations, this cancer is somewhat

more common in males than in females, with a ratio of 1,2 : 1. In terms of worldwide

incidence, colorectal cancer ranks fourth in frequency in men and third in women [4].

Concerning risk for colon cancer, four major categories of risk factors can be identified.

1) Environmental factors. Studies on migrant populations suggest that colon cancer risk is

determined largely by environmental exposure [5]. There are strong geographical differences

which can be denoted to different environmental exposures, i.e. the higher the living standard

and extent of industrialization, the higher the age-adjusted incidence of colorectal cancer [4].

2) Dietary factors. Diet is definitely the most important exogenous factor identified up to now

in the aetiology of colon cancer. There is a strong positive correlation between risk of colon

cancer and per capita consumption patterns of red meat, animal fat and alcohol, whereas a

negative correlation has been reported for vegetable and fibre intake, as shown -after some

controversy- by the large prospective EPIC study [6, 7].

3) Non-dietary factors. Physical activity and chronic use of NSAID’s has consistently been

associated with a decreased colon cancer risk, whereas tobacco use and a BMI above 30

accounts for an increased risk of colon cancer [6].

4) Genetic factors. Family history definitely plays an important role as a risk factor for

colorectal cancer. Interestingly, the molecular basis for malignant colorectal cancers is

relatively well established, at least in comparison with most other human cancers [5]. This

genetic basis is outlined in the next chapter.

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1.2. Genetic basis and pathogenesis

The great majority (> 95 %) of colon cancers is sporadic, but a small tumour subpopulation

arises as a consequence of inherited alleles that create a substantial lifelong risk for this

disease. The colorectal cancers caused by highly penetrating mutations are: Familiar

adenomatous polyposis (FAP) and Hereditary nonpolyposis colorectal cancer (HNPCC) [8].

• FAP defines a condition where more than 100 polyps can be found within one individual,

or less than 100 polyps in an individual with first degree relative with FAP. The frequency

is 1 % of all colon cancer patients. The causal mutation is located on chromosome 5

(5q21-22), affecting the APC tumour suppressor gene. Of all FAP patients nearly 90 %

possess APC mutations. If the gatekeeper mutation is present there is a penetration rate of

nearly 100 %. More than 95% of these mutations, typically insertions or deletions, will

lead to a truncated protein or nonsense mutations [9, 10]. The APC protein is active in the

Wnt signalling pathway; it carries out the phosphorylation of β-catenin, causing its

subsequent proteosomal degradation. This intracellular protein can interact with the

cellular adhesion molecule E-cadherin, which in turn interacts with the actin cytoskeleton

[11]. APC mutations cause cytoplasmic β-catenin accumulation, leading to increased

DNA binding of TCF transcription factors (T Cell Factors) or Lymphoid Enhancer Factors

(LEF), and modulating the expression of the TCF/LEF responsive genes, including genes

involved in proliferation, differentiation, migration and apoptosis (e.g. c-myc). APC also

plays an important role in cell cycle control by inhibiting the G0/G1 to S phase progression

and by stabilising microtubules, thereby promoting chromosomal stability [12]. Figure 1

illustrates how APC mutations can cause polyp formation.

• HNPCC (or Lynch syndrome), is associated with germ line mutations in 6 DNA mismatch

repair genes (MMR) [5]. Several criteria allow diagnosing HNPCC. The frequency is at

least 4% of all colon cancer patients. In 70 % of the cases mutations in MLH1 (3p21),

MSH2 (2p21) or MSH6 (2p16) genes, encoding MMR proteins, are present, with a

penetration rate of 80 % [13,14]. MMR genes function as tumour suppressors.

Homozygous mutations cause a marked decrease in DNA repair, which in turn causes

higher mutation rates (mutator phenotype). MMR mutations also cause microsatellite

instability (MSI), resulting in increased genomic instability [15].

4

Fig. 1| β-catenin and the biology of colonic crypts. Obtained from [16].

Colon cancer has provided an useful model for the understanding of the multistep process of

carcinogenesis ‘thanks’ to the existence of causative mutations as described above.

Considering the Knudson two-hit hypothesis, a progression to a malign carcinoma is more

likely to occur in cells already carrying germ line mutations in APC or MMR genes, in

comparison with non-mutated colonic epithelial cells [17]. Furthermore, both these hereditary

syndromes, as well as sporadic colon cancers, undergo a specific stepwise progression,

described by Vogelstein, starting from normal bowel epithelium and ending in a metastatic

carcinoma (Figure 2). Vogelstein et al. examined genetic alterations in colon cancer

specimens at various stages of neoplastic development and found that changes in the 5q

chromosome, in APC and in the KRAS oncogene tend to occur relatively early in the pathway

[18]. Further downstream in the progression to malignancy is the deletion of chromosome 18.

This is frequently deleted in carcinomas and advanced adenomas and is thus named ‘deleted

in colon cancer’ (DCC). Other mutations, including p53, epigenetic changes such as

methylation of CpG islands of the MLH1 promoter and subsequent mismatch repair defects

lead ultimately towards a malignancy. So there is a progressive acquisition of abnormalities

(over various time-frames) of the genome, affecting known proto-oncogenes or tumour-

suppressor genes and including epigenetic changes, ultimately leading to a metastatic,

invasive carcinoma [6].

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1.3. Therapeutic strategies

Next to radiation therapy and surgery, (poly-)chemotherapy is commonly used and is outlined

in following chapters.

1.3.1. Oxaliplatin

Introduction

Oxaliplatin (OxPt) is a diaminocyclohexane-containing platinum (DACH-Pt) derivative, that

has been approved by the FDA and is widely used in cancer chemotherapy, usually as part of

combination therapies (Figure 3). The DACH-Pt complex of oxaliplatin can exist as three

isomeric conformations that interact differently with DNA, the trans I (R,R) isomer being the

most effective [19].

This third generation platinum anticancer drug was derived from cisplatin (i.e. first

generation) in a screen aimed at identifying platinum analogs with a broader spectrum of

activity, less prone to encounter resistance and with lower (neuro)toxicity than the parent

compound. Accordingly nowadays oxaliplatin is currently used in the clinical management of

cisplatin-resistant tumours, including colorectal cancers, that are intrinsically resistant to

cisplatin [20, 21].

Fig. 2| Vogelstein model for the carcinogenesis of colon cancer. Obtained and adapted from [18].

Fig. 3| Chemical structure of Oxaliplatin:

1,2-diaminocyclohexaneoxalato platinum.

Obtained and adapted from [20].

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Mechanism of action

OxPt enters the cell by passive diffusion and with the help of high-affinity copper transporters

(CTR1). Hereafter the molecule is converted into an aquated form that is more reactive than

the parent molecule, especially towards DNA, with which it forms different types of adducts

(Figure 4).

These adducts arise because the platinum atom of oxaliplatin forms covalent bonds to the N7

positions of purine bases -preferably of nuclear DNA- resulting primarily in 1,2- or 1,3-

intrastrand crosslinks. In addition, interstrand crosslinks and DNA-protein crosslinks have

also been reported, although with lower frequency. DNA binding can occur by displacement

of the oxalate ligands originally present in the compound [19, 20]. Because of the steric

hindrance of the DACH carrier group, oxaliplatin distorts the DNA duplex, bending it

significantly towards the major groove, which in turn exposes a wide, shallow minor groove

surface to which several classes of proteins can bind and activate several cellular processes

that mediate the cytotoxic effect of this drug [21]. These (interfering) binding proteins are:

• Mismatch repair proteins. The binding of the mismatch repair complex to Pt–DNA

adducts appears to increase the cytotoxicity. This either by activating downstream

signalling pathways that lead to apoptosis or by causing ‘futile cycling’ during

translesion synthesis past Pt–DNA adducts. Although this would seem logic for all Pt-

DNA adducts, these effects appear to be specific for cisplatin but not for OxPt

adducts; MMR deficiency or MMR mutations (hMSH2 and MutS, both components of

the MMR complex) are apparently not a determining factor for the OxPt treatment

outcome [22] .

• Damage recognition proteins such as: i) HMG box proteins (e.g. structure-specific

recognition protein 1 (SRP1) or high-mobility group box protein 1 (HMGB1) as the

most abundant ones), ii) TATA box-binding proteins (TBP) and iii) human upstream

binding factors (UBF). HMGB1 has been linked to several DNA-dependent pathways

Fig. 4| Biotransformation pathway of Oxaliplatin. Obtained from [19].

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(i.e. RAG1/2, MAPK and p53 possibly leading to apoptosis via a Bax-dependent

pathway) and modulates the efficiency of nucleotide excision repair [20].

So the DNA damage caused by OxPt modulates several signal transduction pathways (e.g.

AKT, c-ABL, p53, p38MAPK, JNK and ERK pathways) finally determining the cytotoxic

and/or resistance outcome. Next to these pathways, DNA-Pt adducts can also directly block

DNA replication and transcription [20].

DNA is not the only target for OxPt, cellular proteins may also be affected. Knowing that 75-

85 % of the intracellular OxPt is bound to proteins, it is not surprising that this effect can also

lead to detrimental consequences. Meynard et. al. postulated in 2007 the following

hypothesis. “After entry in the cell, oxaliplatin would especially target the thiol groups (i.e.

cysteine and methionine) of nascent proteins, which would be particularly sensitive to the

reactive oxygen species (ROS) produced by mitochondrial respiration. The resulting protein

oxidation would be at the origin of cell death.” If this hypothesis is correct, both DNA and

protein-mediated damage could lead to apoptosis in response to oxaliplatin treatment [23].

Resistance mechanisms

Some of the resistance mechanisms counteracting OxPt cytotoxicity are drug-specific, while

others more generally affect other drugs and/or act in a cell-type specific fashion (see

Discussion).

• Translesion (TLS) DNA polymerases. Several translesion DNA polymerases have

been shown to bypass Pt–GG intrastrand adducts. TLS polymerases can bypass the

damage in an error-free or error-prone fashion, the first resulting in a resistance

mechanism, the latter in elevated mutagenesis, possibly leading to apoptosis [22].

• Nucleotide excision repair (NER). NER is the only known mechanism by which bulky

adducts (as those caused by DACH) are removed from DNA in human cells; increased

activity of this repair system is one of the major causes of OxPt resistance [24].

• Nonspecific inactivation of OxPt [20].

• Decreased expression of the copper influx transporter CTR1, and/or, increased

expression of the copper efflux transporter ATP7A mediates OxPt resistance [25].

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1.3.2. 5-Fluorouracil

Introduction

5-Fluorouracil (5FU) is widely used in the treatment of cancer. Despite the fact that the

mechanisms of action of this drug are clearly understood, resistance remains a significant

limitation to the clinical use of 5FU. This fluoropyrimidine drug has a dual function in

inhibiting the normal cellular metabolism. First, 5FU is an uracil analog in which a fluorine

atom replaces hydrogen at the C-5 position; following activation and phosphorylation to the

corresponding triphosphate nucleotide the drug is misincorporated into RNA end DNA,

disrupting nucleotide-synthesis. Second, 5FU inhibits the nucleotide synthetic enzyme

thymidylate synthase (TS). It might be important to note that in vivo more than 80% of

administered 5FU is normally catabolised primarily in the liver, where dihydropyrimidine

dehydrogenase (DPD) is abundantly expressed (this is obviously not the case in our in vitro

experiments).

Modulation strategies, such as co-treatment with leucovorin and methotrexate, have been

developed to increase the anticancer activity of 5FU. Response rates to 5FU in advanced

colorectal cancer have been dramatically improved by combining the drug with OxPt and

irinotecan (See further) [26].

Mechanism of action

After facilitated transport into the cell, 5FU is converted intracellular into several active

metabolites, that are responsible for the cytotoxic activity of the compound:

fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and

fluorouridine triphosphate (FUTP). DPD, converting 5FU into its inactive metabolite

dihydrofluoruracil (DHFU), is the rate-limiting enzyme in 5FU catabolism. See Figure 5 for

an overview.

9

Normally, TS catalyses the conversion of deoxyuridine monophosphate (dUMP) to

deoxythymidine monophosphate (dTMP) with 5,10- methylene tetrahydrofolate (CH2THF) as

the methyl donor. This reaction is the only de novo source of thymidylate. In contrast, when

5FU is present, the active metabolite FdUMP binds to the nucleotide-binding site of TS and

forms a stable ternary complex with TS and CH2THF, thereby blocking the access of dUMP

to the nucleotide-binding site and inhibiting dTMP synthesis. This ultimately results in

deoxynucleotide (dNTP) pool imbalance and increased levels of dexouridine triphosphate

(dUTP), both of which cause DNA damage through a disruptive DNA synthesis and repair

[26]. See figure 6 for an overview.

Fig. 5| 5-Fluorouracil metabolism and chemical structure (Left under). Obtained from [26].

Fig. 6| Mechanism of thymidylate synthase inhibition by 5FU. Obtained from [26].

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Resistance mechanisms

Resistance is a major obstacle to the success of 5FU-based therapies. Some resistance

mechanisms specific for 5FU are listed below.

• dTMP can be salvaged from thymidine through the action of thymidine kinase (TK),

thereby alleviating the effects of TS deficiency; this represents a potential resistance

mechanism [26].

• Treatment with 5FU has been shown to acutely induce TS expression. This induction

seems to be the result of inhibition of a negative-feedback mechanism in which

ligand-free TS protein binds to, and inhibits the translation of TS mRNA. When stably

bound by FdUMP, TS is no longer able to bind to its mRNA and suppress its own

translation, resulting in increased TS protein expression [26].

• In vitro studies have shown that DPD overexpression in cancer cell lines confers

resistance to 5FU. Furthermore, high levels of DPD mRNA expression in colon

tumours have been shown to correlate with resistance [27].

• Overexpression of p53 correlates with resistance to 5FU, although in vitro studies

reported that loss of function of p53 also reduces cellular sensitivity to 5FU [28].

• Expression levels of mRNA’s encoding the multidrug resistance proteins MDR3/4

were found to significantly correlate with 5FU sensitivity [26].

• Cells possessing MMR defects (i.e. MLH1-), which is often the case in colon cancer

(particularly in HNPCC), and which causes MSI have been found to be 18-fold more

resistant to 5FU than cells with normally functioning MMR [29]. However, the MSI

phenotype has been associated with excellent survival in patients who receive

adjuvant 5FU based chemotherapy. These contradictory findings can possibly be

explained by intrinsic biological differences between MSI+ (e.g. p53 wt) and MSI

-

(e.g. p53 mutated) tumours [26, 30].

DNA microarray analysis of 5FU-responsive genes playing key roles in resistance will

facilitate the identification of new biomarkers and rational drug combinations. Some

molecular biomarkers that predict tumour sensitivity to 5FU have already been identified,

including mRNA and protein expression levels of TS, indicating that patient selection based

on the molecular profile of their individual tumours might help increase the response rate to

5FU treatment.

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1.3.3. Cetuximab & Bevacizumab

Because conventional cytotoxic agents, including OxPt and 5FU are administered

systemically, they will affect not only tumour cells, but also normal proliferating cells in the

organism, so that side toxicities are major limiting factors to chemotherapy [31]. An ideal

approach would be to identify pathways that are exclusively altered in tumour cells and to

target them selectively (target-driven therapy) however, this is rarely the case. Although,

context-driven therapies, based on cell intrinsic or extrinsic differences that cause tumour

cells to rely on a specific pathway more than normal cells, represents a way to reduce side

effects. Antibodies have been developed to selectively target a tumour cells based on

quantitative differences in the expression of specific surface antigens, e.g. growth factor

receptors [32]. At present, two monoclonal antibodies have been approved by the FDA for use

in metastatic colon cancer:

• Cetuximab (Erbitux) is a chimeric monoclonal IgG1 antibody that acts by binding to

the extracellular domain of the Epidermal Growth Factor receptor (EGFR, belonging

to the ErbB family), preventing ligand binding and receptor activation, thereby

blocking the signalling downstream of EGFR and resulting in impaired cell growth

and proliferation. Cetuximab also mediates ADCC (Antibody-Dependent Cellular

Cytotoxicity) [31].

• Bevacizumab (Avastin) is a humanised monoclonal IgG1 antibody that binds the pro-

angiogenic factor VEGF (Vascular Endothelial Growth Factor) and prevents its

binding to specific tyrosine kinase receptors, inhibiting angiogenesis [33].

1.3.4. Combination strategies

To achieve an optimal clinical outcome several combination strategies with synergistic

cytotoxic effects have been developed, hence 4 regimens can be distinguished these days.

Dependent on the grade of the colon cancer (grade I, IIA, IIB, IIIA, IIIB, IIIC or IV) one

regimen can be chosen and variations in chemotherapy dosing and time schedules are

possible. With the aid of these different chemotherapy regimens an pursuance towards an

optimal and fine-tuned balance between toxicity and resistance is possible [34]. The four most

common regimens are the following, each one apparently causing specific side effects:

12

1. Fluoropyrimidine based:

• LV5FU2: 5FU and Leucovorin (LV). LV or folinic acid is a reduced

folate that is thought to stabilize fluorouracil’s interaction with

thymidylate synthase. This modulation doubles the response rate with

a statistically significant improvement in disease-free and overall

survival of patients with metastatic colon cancer compared with 5FU

alone [35].

• There are several hospital-specific (dose) modulations.

2. Oxaliplatin based:

• FOLFOX: 5FU, LV and oxaliplatin [36].

• Modified FOLFOX and FLOX: Modulations concerning the dose and

way of administering (bolus/infusion).

3. Irinotecan based:

• FOLFIRI: 5FU, LV and irinotecan. Irinotecan is a semisynthetic

derivative of the natural alkaloid camptothecin and inhibits

topoisomerase I, an enzyme that catalyzes breakage and rejoining of

DNA strands during DNA replication [37].

• IFL: Modulations concerning the dose and way of administering [34].

4. Antibody based:

• Cetuximab + irinotecan and/or 5FU [34].

• Bevacizumab + irinotecan and/or 5FU [33].

2. Colon cancer & Hypoxia

2.1. Hypoxia

Hypoxia can be defined as a state of reduced O2 availability or decreased O2 partial pressure

below critical thresholds, thus restricting or even abolishing the function of organs, tissues, or

cells. There is a clear evidence that these hypoxic thresholds can vary widely, although an

upper limit of 35 mmHg can be set [38]. Relative low partial oxygen pressure is a common

feature in many solid tumours, including colon carcinomas. Because of the rapid proliferation

of the tumour mass and the limited diffusion distance (i.e. 100-200 µm; dependent on the

vasculature), cancer cells become hypoxic as they outgrow the standard blood supply [39].

Tumour hypoxia is a powerful driving force for malignant progression and has been identified

13

as an adverse prognostic factor, as clinical and preclinical studies have firmly established that

hypoxia is associated with impaired response to both radiotherapy and chemotherapy [38, 40].

This latter effect is due in part to poor perfusion and restricted drug access to hypoxic areas,

but hypoxia-dependent adaptive changes in gene expression probably play the major role in

reduced drug response [41]. Hypoxic stress, induced by a decrease in O2 partial pressure

below 5% (40 mmHg) activates the transcription factor Hypoxia-inducible factor 1 (HIF-1), a

heterodimer composed of an inducible, oxygen-sensitive α subunit and a constitutively

expressed β subunit, that is considered as the master regulator of the hypoxic world (Figure 7)

[42].

Overall, HIF-1 is responsible for the transcription of more than 100 putative genes (HRE’s;

Hypoxia Responsive Elements) in hypoxic circumstances and this occurs not only in

cancerogenesis, but also during normal development and several pathophysiologic conditions.

[42, 43]. See Figure 8 for an overview.

Fig. 7| Degradation and activation of the

HIF-1α transcription factor in normoxia

and hypoxia. Obtained from [42].

Fig. 8| Genes that are transcriptionally

activated by HIF-1. Obtained from [43].

14

The major HIF-1 dependent pathways are involved in the control of:

• Cellular proliferation: hypoxia induces the expression of growth factors stimulating

cell proliferation, such as PDGF, TGF-α and IGF2 [44].

• Metabolism: under hypoxic conditions a switch occurs from aerobic metabolism to

anaerobic glycolysis. HIF-1 regulates the expression of enzymes that are necessary for

the glycolysis and glucose transporters (GLUT1 and GLUT3, mediating glucose

uptake by the cells) [45].

• Angiogenesis: HIF-1 activates angiogenesis, via enhanced expression of the vascular

growth factors ANG2 (angiopoeitin-2) and VEGF, encoded by one of the best known

HIF-1 target genes [46].

• Apoptosis: under hypoxic conditions apoptosis is induced following HIF-1-dependent

accumulation of p53, which results in selection of cells carrying mutations in p53

and/or other genes involved in apoptosis control, making tumour cells less prone to

drug-induced cell death. In addition, HIF-1 has been shown to negatively regulate the

expression of pro-apoptotic genes in human colon cancer cells, thereby shifting the

balance even further towards cell survival [47].

• Immortality: under hypoxic circumstances an increase is observed in telomerase

activity, an enzyme essential to sustain the unlimited proliferative potential of tumour

cells [48].

• pH regulation: hypoxia-induced activation of anaerobic glycolysis and increased

expression of type IX carbonic anhydrase results in production of lactic acid and

carbon dioxide, respectively, both causing intracellular acidification relative to the

extracellular space. This in turn contributes to tumour invasion by activating a number

of proteases dependent on acidic pH (see further). HIF-1 has been shown to regulate

invasive behaviour in HCC (Human colon carcinoma) cells [49]. So hypoxic cells -in

contrast to normal cells- are able to escape and thus survive from a relatively acidic

microenvironment [50].

• Drug resistance: HIF-1 has been shown to induce mdr1 gene expression (Multidrug

Resistance gene 1 or Glycoprotein-P) enhancing drug efflux [51].

The activation of one or more of these pathways is a substantial advantage for the fast

growing tumour mass, and indeed, overexpression of the HIF-1α subunit has been

demonstrated in colorectal cancers [52].

15

2.2. HIF-1 structure and regulation

HIF-1 is the most significant and best studied member of a group of hypoxia inducible factors

(HIFs) and is composed of an oxygen-sensitive HIF-1α subunit and a constitutively expressed

HIF-1β subunit, also known as aryl hydrocarbon nuclear translocator (ARNT). The 2 subunits

have a similar domain structure and they contain a basic helix-loop-helix domain, required for

their dimerization and DNA binding, a Per-ARNT-Sim domain (PAS), that also is important

for dimer formation, and a transactivation domain (TAD). The TAD domain of HIF-1α that

can be subdivided in N-TAD and C-TAD, and has been shown to bind the co-activator

proteins p300/CBP, SRC-1 and TIF2, whereas the TAD of HIF-1β appears to be dispensable

for the activity of the HIF-1 complex [53]. The HIF-1α gene promoter contains recognition

sites for several ubiquitous transcriptional activators, such as Sp-1, AP-1, AP-2 and NF-1,

causing the gene to be constitutively expressed; however, in normal cells under normoxic

conditions HIF-1α is undetectable, due to fast protein degradation. HIF-1α is a 826 amino

acid protein with a molecular weight of 120 kDa [50].

2.2.1. Oxygen dependent HIF-1 regulation

Under normoxic conditions, HIF-1α becomes hydroxylated on two proline residues (402 and

564, located in the so-called oxygen dependent degradation domain, ODDD, overlapped by

N-TAD) by a family of prolyl hydroxylases (PHD1-3) [54]. Due to this hydroxylation, the

von Hippel-Lindau protein (pVHL), a recognition component of an E3 ubiquitin ligase,

recognises the HIF-1α subunit, targeting it for polyubiquitylation and subsequent degradation

of HIF-1α by the 26S proteosomal system. Another so called oxygen sensor is Factor

inhibiting HIF-1 (FIH-1), an oxygen-dependent enzyme that hydroxylates Asn803 within the

C-TAD of HIF-1α, disrupting its interaction with the transcriptional co-activators p300 and

CBP. Thus, the two types of metabolic sensors, PHDs and FIH, by controlling both the

destruction and inactivation of HIF-α subunits, ensure full repression of the HIF pathway in

well-oxygenated cells (Figure 9) [42].

16

These 2 oxygen sensors possess a different oxygen affinity hence a fine tuning of

transcription of HREs is possible; PHD has a much lower affinity for oxygen than FIH, so

that PHD activity is decreased under moderate hypoxic conditions and inactive under

complete hypoxia, whereas severe hypoxia is required to inactivate [50]. This observation,

together with the fact that PHDs cause HIF-1α, thereby silencing both N-TAD and C-TAD

genes, whereas FIH only targets the C-TAD of the HIF-1α protein, can explain the

‘bicephalous’ transcriptional nature of HIF-1α and its ability to differentially regulate two sets

of genes (i.e. the N-TAD and C-TAD genes) (Figure 10).

Fig. 9| Oxygen sensors contribute to the destruction and inactivation of HIF-1α. Obtained from [50].

Fig. 10| Working model of two sets of HIF-1 regulated genes. The further away from blood vessels, the

more hypoxic the cells and the higher the extracellular acidity due to the accumulation of lactate and

CO2. Hypoxia also induces the expression of carbonic anhydrase IX (CA IX), which helps to retain a

relatively neutral intracellular pH, furthermore there is an expression of the proapoptotic protein BNIP-3

under moderately hypoxic conditions, but requires acidosis to promote cell death which occurs in

extreme low pO2 conditions. Obtained from [50].

17

An extra fine-tuning of specific gene activation by HIF may result from isoform specificity,

because three isoforms of HIF-α and several splice variants of each exist and increasing

evidence suggests that specific genes may be activated by one or the other or several

isoforms. Further regulation of HIF-1α is ensured by other post-translational modifications

such as: phosphorylation of HIF-1α, which enhances transcriptional activity, and interaction

with heat shock protein 90 (Hsp90), which regulates its stability [42].

Under hypoxic conditions, the above described degradation and inactivation will not happen

and dimerisation between the HIF-1α and HIF-1β subunit occurs. Together they bind to

hypoxia-response elements (HRE’s) throughout the genome, recruiting transcriptional co-

activators and upregulating target gene expression [16].

2.2.2. Oxygen independent HIF-1 regulation

Diverse stimuli including growth factors, cytokines, NO or oncogene activation can activate

HIF-1 under normoxic conditions through the phosphatidylinositol 3-kinase (PI3K) and

mitogen-activated protein kinase (MAPK) pathways in a cell-type-specific manner (whereas

oxygen-dependent HIF-1 regulation occurs in every cell type) (Figure 11) [43]. The most

notable growth factors herein are insulin-like growth factor-2 (IGF2) and transforming growth

factor-α (TGF-α), encoded by HIF-1 target genes itself, thus creating an autocrine-signalling

pathway when binding to their cognate receptors (IGF1R and EGFR, respectively) and

activating signal-transduction pathways that lead to HIF-1α expression and cell

proliferation/survival, both crucial for cancer progression [49]. An improved stabilisation of

HIF-1α in normoxic circumstances can be achieved by activation of oncogenes such as Src or

Ras, and/or inactivation of the tumour supressor genes such as PTEN and VHL. All this

results in an substantial increase in translation of the HIF-1α mRNA through phosphorylation

of the eukaryotic translation initiation factor 4E (eIF-4E), which finally results in HIF-1

protein expression, which is particular sensitive to changes of synthesis-velocity because of

its extremely short half-life in normoxic conditions (< 5 min) [50].

18

2.3. HIF-1 and drug resistance

Besides the features described above, that concur to tumor progression under hypoxic

conditions, HIF-1 or hypoxia can also contribute to the development of drug resistance in

several ways:

• Downregulation of the pro-apoptotic proteins Bid and Bax [47].

• Direct upregulation of Bcl-xL [55].

• Downregulation of DNA repair proteins [56].

• HIF-1 promotes the formation of an ‘aggressive’ and abnormal vasculature, which has

an negative influence on drug delivery to the tumour.

• Due to the acidification, some drugs are retained less efficiently inside the cell and/or

are less cytotoxic, impairing their effectiveness [57].

• Hypoxia is also associated with resistance to X-Ray therapy, as this therapeutic

modality relies on reactive oxygen species that cannot be efficiently produced under

hypoxic conditions [43].

Fig. 11| Regulation of O2 independent HIF-1 protein synthesis. Obtained from [43].

19

Thus, selection by hypoxia may explain the resistance of many solid tumours not only to

hypoxia-induced-apoptosis, but to radio and chemotherapy as well. Furthermore, through this

selection these cells increase their potential for invasion and metastasis, thereby considerably

worsening patient prognosis [38].

Importantly, HIF-1-dependent tumour cell response can be affected by the p53 tumour

suppressor gene status [58]. After prolonged exposure to hypoxic conditions, p53

downregulates HIF-1α and also the transactivating function of HIF-1α is repressed because of

the competition for the co-activator p300 [59]. This implies that p53 deletion or loss of

function mutations, that are extremely common in tumours derived from epithelial tissues,

including colon carcinomas, will also have an impact on HIF-1α activity, promoting

angiogenesis and other pro-tumour activities [58, 60]. Loss of p53 function may also directly

contribute to drug resistance [61].

From these observations, it can be concluded that induction of HIF-1α and HIF-1 activation

may play a major role in the resistance of hypoxic tumour cells to killing by chemotherapy (or

radiation). Thus, it will be important to determine whether HIF-1α alone or trough crosstalk

with other markers such as p53 can be seen as a valid marker for chemoresistance. Based on

these and other considerations, targeting HIF-1α or HIF pathways may represent an attractive

strategy to potentiate the antitumour effects of conventional cytotoxic agents in colon cancer.

3. Modulation of HIF-1

Based on the multiple roles played in tumour progression and tumour drug response, HIF-1

has become an important therapeutic target. Many compounds already known to act on other

cellular mechanisms or signalling pathways (e.g. including the topoisomerase I inhibitor

topotecan, the natural phytoalexin resveratrol and the guanylyl cyclase activator YC-1) have

been shown to affect HIF-1 and others have been developed or are under development that

target HIF-1, either directly or via modulation of HIF-1α levels [62, 63, 64]. In the present

study, we have used the small molecule inhibitor of thioredoxin PMX290 and the locked

nucleic acid antisense oligonucleotide, described below.

20

Fig. 13| L.N.A.’s with a clearly

visible CH3 bridge between

2’-O and 4’-C atom. Obtained

from [67].

3.1. PMX290

PMX290 is a small molecule inhibitor of the

thioredoxin system (Figure 12). The thioredoxin (Trx)

system includes Trx1 and 2, two low molecular weight

proteins containing thiol (SH) that are oxidized while

providing reducing equivalents to target molecules e.g.

ribonucleotide reductase (which is involved in DNA

synthesis), peroxiredoxin (is a cellular antioxidants) and

various transcription factors. Trx’s are then recycled by

Trx reductase (TrxR) in a NADPH (nicotinamide

adenine dinucleotiede phosphate-oxidase)-dependent reaction. Trx1 is the predominant form

and is localised in the cytosol whereas Trx2 has been identified in mitochondria and executes

functions of the electron-transport chain [65]. It is already apparent that Trx-1 is upregulated

in hypoxic regions of solid tumours, where it is hypothesized to cause HIF-1 activation and to

regulate vascular endothelial growth factor levels and hence angiogenesis. Inhibiting Trx-1

function using PMX290 has been shown to impair HIF-1α CAD transcription activity and

DNA binding; therefore, this compound has been used in this study to verify the role of HIF-1

in drug resistance and to sensitize colon cancer cells to the effects of cytotoxic drugs [66].

3.2. Antisense oligonucleotides

HIF-1α expression was knocked down by transfection with the

antisense oligonucleotide EZN-2968 directed against HIF-1α

mRNA. The antisense sequence is 5’-TGGcaagcatccTGTa-3’

where lower case letters represent “natural”

deoxyribonucleotide residues, whereas capital letters represent

nucleotides featuring a Locked Nucleic Acid (LNA) structure,

with the ribose ring is “locked” by a methylene bridge

connecting the 2’-O atom and the 4’-C atom (Figure 13). LNA

structure is ideally suited for Watson-Crick base pairing and

pairing with a complementary nucleotide strand is more rapid

and the resulting duplex exhibits increased thermal stability as

compared to native oligonucleotide sequences [67].

Fig. 12| Chemical structure of PMX -

290 (=AJM290). Obtained from [66].

21

The sequence is also relatively resistant to exo-and endonucleases, due to replacement of

phosphodiester with phosphorothioate internucleotide bonds, and exhibits a high target

specificity. EZN-2968 is complementary to nucleotides 1197 to 1212 in the human HIF-1α

mRNA sequence [68].

4. Aims

I. How does hypoxia affect the cellular response to oxaliplatin and 5FU?

To address this question, HCT116 and HT29 cells were grown as monolayers and exposed to

a range of oxaliplatin and 5FU concentrations under normoxic and hypoxic conditions. At this

stage, the role of HIF-1 in tumour cell response to the drug is not directly addressed, as

hypoxia is known to induce a pleiotropic response that depends for the most part on HIF-1

activation, but that can also involve HIF-1-independent mechanisms. In addition to this

model, HCT116 cells were grown as 3D spheroids. In this case hypoxia will develop

spontaneously once the spheroid size exceeds 100-200 µm in diameter, so the response to

5FU will be compared with the response of normoxic cells grown as monolayers. Drug

sensitivity was assessed by the following methods and results are presented as IC50 values or

apoptotic cell percentages:

1. Cell counting (effect on cell growth and survival)

2. Clonogenic assay (effect on the clonogenic potential)

3. FACS analysis of cells stained with propidium iodide (cytotoxic effect)

II. Is HIF-1 activity directly involved in tumour cell response under hypoxic conditions?

To explain the results obtained in aim I, HIF-1 transcriptional activity was determined by

method of HCT116 transfection with a plasmid containing EGFP cDNA under the control of

an hypoxia-responsive promoter (HRP) and subsequent analysis by FACS. HIF-1

transcriptional activity is expressed as EGFP fluorescence and was compared between

HCT116 spheroids and HCT116 monolayers.

22

III. Is it possible to modify colon cancer cell response to OxPt and 5FU by modulating the

expression of HIF-1α or HIF-1 transcriptional activity, independent of oxygen levels?

This question was addressed by setting up different experimental models whereby HIF-1α

expression or HIF-1 transcriptional activity can be positively or negatively modulated,

irrespective of oxygen conditions. To this aim, we have used HIF-1 modulating agents

(PMX290, oligonucleotides and HIF-1αMUT) and this way we were finally able to assess the

altered effect of 5FU and OxPt in a quantitative and qualitative manner. Results are presented

as apoptotic cell percentages, IC50 values and EGFP fluorescences.

IV. Does p53 status significantly influence the response of colon cancer cells to HIF-1

manipulations?

We have proceeded as described in aims I, II and III, but using the HT29 cell line, expressing

a mutant form of p53. The presence of mutant p53 could lead to a different response to OxPt

or 5FU under normoxic and/or hypoxic conditions as compared to p53wt cells and could

interfere with HIF-1 dependent processes. This issue was investigated by comparing the

effects of HIF-1α manipulation (up- or downregulation) in HT29 cells with those observed in

p53 wt-bearing HCT116 cells. The same methods applied to HCT116 were used.

Thus, the general aim of this project is to assess the effects of HIF-1 modulation

through novel HIF modulating factors, on the response of cultured colon cancer cells

to different cytotoxic agents currently used against colon cancer, namely 5FU and

oxaliplatin. Because of the described crosstalk between HIF-1 and p53, cell lines

characterized by different p53 status were used. HIF modulators might represent an

important, novel approach to circumvent chemoresistance in colon cancer.

The research activities described in this project were performed in the laboratory of anticancer

pharmacology, University of Insubria, Busto Arsizio, Italy, where the main research focus is

currently on the molecular mechanisms of tumour cell response (or the lack thereof) to

combinations of conventional cytotoxic drugs with novel targeted antitumour approaches,

using in vitro and in vivo models.

23

Materials & Methods

Cell lines

We have used different human colon cancer cell lines (Obtained from A.T.C.C., Rockville,

MD);

� HT29: Human colon cancer cell line isolated from an adult female Caucasian patient.

These cells express an inactive mutant of the p53 tumour suppressor protein (R237H)

bear a mutation in the APC gene, but do not have defects in the mismatch repair

system [69]. They display an epithelial morphology.

� HCT116: Human colon cancer cell line derived from an adult male patient. They bear

a p53 wild type gene. The HCT116 cells have an activating mutation in K-Ras and

bear a defect in the mismatch repair system [69]. They appear as spindle shaped cells

with an epithelial morphology.

� HCT116/HRP-EGFP cells were obtained from HCT116 cells by transfection with a

plasmid containing the EGFP (enhanced green fluorescent protein) cDNA under the

control of an artificial hypoxia-responsive

promoter (HRP) consisting of five copies of a

35-bp fragment from the HRE of the human

VEGF gene and a human cytomegalovirus

(CMV) minimal promoter (kindly provided by

Dr. Y. Cao) [70]. Figure 14 shows the

structure of this plasmid. The presence of the

neomycin-resistance gene (Neor) allowed

selection of stably transfected clones after 2

weeks of growth in media containing 500

µg/ml of the G418 antibiotic; the clone with the highest EGFP induction was used.

Cell cultures

Cells were grown in Dulbecco Modified Eagle’s Medium (DMEM) in the case of HCT116,

whereas for HT29 cells we used McCoy’s Medium, both supplemented with 10 % fetal

bovine serum (EuroClone, Italy), 1 % antibiotic mixture (penicillin/streptomycin), 1 % non

Fig. 14| Plasmid used for transfection of

HCT116 cells to obtain HCT116/HRP-

EGFP cells.

24

essential aminoacids and 1 % L-glutamine, all maintained in 37 °C in a humidified

atmosphere. Cells were incubated in either normoxic (48 h 21 % pO2, 5% pCO2, 74% pN2 ) or

where appropriate in hypoxic conditions (24 h normoxia, subsequently 24 h 1% pO2, 5%

pCO2, 94% pN2). This was achieved by placing the cells in a modular incubation chamber

(Billups-Rothenberg Inc., Del Mar, CA, USA), during the last 24 h.

Three-dimensional spheroids

In contrast with monolayer cultures, multicellular spheroids spontaneously develop hypoxic

areas, thereby reproducing more closely the in vivo situation. To obtain three-dimensional

spheroids, HCT116, HCT116/HRP-EGFP and HT29 cells, grown as monolayers, were

detached by trypsinization and subsequently seeded (5 x 103 cells/well) onto 96-well tissue

culture plates coated with 1,5 % agarose to prevent cell attachment. Complete medium was

used, supplemented with an 1 % extra sodium pyruvate to achieve a better growth of the cells

as spheroids. Cells were incubated at 37 °C in a humidified 5% CO2 atmosphere and grown

for 7 days: at the end of this period of time spheroids are formed, reaching an average

diameter of 600 µm and consisting of approximately 5000 cells (see Figure 18 in the Results

section). Seven-day spheroids were used for flow cytometric and cytotoxicity studies.

Drug treatments

For all the experiments, cells were exposed to chemotherapeutic drugs, either 5FU or OxPt,

with or without HIF-1-modulating agents. PMX290 (formerly AJM290, kindly provided by

Prof. M.F. Stevens, University of Nottingham and Pharminox Ltd., UK) was used at various

concentrations for 48 h. For this HIF-1 modulator stock solutions were prepared in DMSO

(10 mM for PMX290); cellular exposure to DMSO never exceeded 0,025 %.

Transfection with antisense oligonucleotides

Cells were transfected with EZN-2968 (described in the Introduction section); a scrambled

oligonucleotide (EZN-3088) with the sequence 5’-CGTcagtatgcgAATc-3’ was used as

control for non sequence-specific effects. Finally, mock-transfection, using the lipofection

reagent without oligonucleotides, was performed on control cells (indicated as MOCK).

HCT116 and HT29 cells were seeded with a concentration of respectively 40.104 cells/ml and

25

15.104 cells/ml and transfection was performed when a 50 % confluence was obtained (after

72 h) in 100 mm/15 mm petri-dishes. After 2 washes with filtered Opti-Mem (Gibco,

Invitrogen) medium without antibiotics and serum, 6 ml of Opti-Mem, containing 5 µg/ml of

lipofection reagent (Lipofectamin2000, Invitrogen), were added to the dishes. After 15’

incubation at 37°C, an additional 1,5 ml of Opti-MEM containing the oligonucleotides at the

final concentrations of 10 and 100 ng/ml were added. After 4 hours of incubation at 37° C,

cells were washed with Opti-MEM and 10 ml of complete medium was added to terminate

the lipofection reaction. Transfected cells were grown for 24 hours, at the end of which they

were detached and used for the different experiments.

Construction of lentiviral vectors (HIF1α-mut)

Lentiviral particles were generated using a transient expression system, composed of (1) the

pCMV∆R8.74 packaging construct, (2) the pMD2.G envelope expression construct and (3) a

task-specific lentiviral vector: the pWPT-GFP transfer vector, for overexpression of a HIF-1α

degradation-resistant mutant cDNA. The plasmids pWPT/GFP contain a green fluorescent

protein (GFP) cDNA under the transcriptional control of an intronless human elongation

factor 1-α (EF1-α-short) promoter. All constructs were kindly provided by Dr. Didier Trono

(School of Life Sciences, Swiss Institute of Technology, Lausanne, Switzerland). The transfer

vector pWPT/HIF-1αMUT/GFP was generated by cloning a 3100-bp fragment containing the

cDNA of a human mutated form of HIF-1α (kindly provided by Dr. Chris Paraskeva,

University of Bristol, UK) into the pWPT/GFP vector. In HIF-1αMUT, Pro 402 and Pro 564

have been replaced by alanine and glycine, respectively; these modifications prevent oxygen-

dependent prolyl hydroxylations, so that the protein is degradation-resistant under normoxic

conditions [71].

Generation of lentiviral particles and target cell infection.

Lentiviral particles pseudotyped with the VSV envelope glycoprotein were produced by co-

transfecting 5.106 293FT cells with 40 µg of total plasmid DNA: the i) pCMV∆R8.74, ii)

pMD2.G and iii) pWPT/HIF-1α MUT vector, with the calcium phosphate precipitation

method, as previously described [72]. Transduction experiments were performed in a medium

containing 4 µg/ml polybrene. Viral titration was performed by flow cytometer-counting

GFP-expressing HCT116 cells 48 h after infection. For in vitro mutant-HIF-1α

26

overexpression experiments, 30 % confluent HCT116 cells were infected for 4 h with 10

MOI lentiviral particles; the particle-containing medium was then replaced with fresh medium

and cells were incubated at 37 ° C for 48 h before use.

Assessment of HIF-1α expression by Western blot analysis.

Western blot analysis was carried out to detect the expression of HIF-1α in whole cell lysates,

following normoxic or hypoxic incubation and/or drug treatment. After harvesting, counting

and centrifugation (1300 rpm, 10’, 4°C), cells were washed with 1 ml of Phosphate Buffer

Saline (PBS). After another centrifugation cells were lysed in a buffer containing NaF 25

mM, EDTA 5 mM, sodium pyrophosphate 25mM in TBS 20 mM, pH 7,4, PMSF 2 mM,

Na3VO4 1 mM, phenylarsine oxide 1 mM, 1% v/v NP-40 and 10 % v/v Protease Inhibitor

Cocktail (Sigma), at the concentration of 100 µl per 107 cells. Subsequently, while holding the

samples in ice, a sonication was performed (2 times 10”, cycle = 1, amplitude % = 100).

Then, a centrifugation 12800 rpm for 20’ was performed and the supernatant was collected.

Protein concentration was determined by the BCA assay (Pierce, Italy) and 100 µg of protein

per sample was loaded onto polyacrylamide gels (8%) and separated under denaturing

conditions. Protein bands were then transferred onto Hybond-P membranes (Amersham

Biosciences, Italy) using Bio-Rad, Trans-Blot SD and Western blot analysis was performed

by standard techniques with mouse monoclonal anti-human HIF-1α antibody (BD,

Biosciences; dilution 1:300). Equal loading of the samples was verified by re-probing the

blots with a mouse monoclonal anti-actin antibody (Santa Cruz Biotechnology Inc.; dilution

1:1000). Protein bands were visualised using a peroxidase-conjugated antimouse secondary

antibody (Sigma-Aldrich; dilution 1:4000) and the Supersignal West Femto Maximum

Sensitivity Substrate (Pierce, Italy).

Effects on cell growth

The antiproliferative effects of 5FU and OxPt were assessed based on cell counts. Cells (i.e.

HCT116, HT29, HCT116 WPT and HCT116/HIF-1α MUT cells) (3,5.103/well) were seeded

onto 24-well plates, allowed to attach and grow for 24 h and subsequently exposed to

different 5FU (1-250 µM) and OxPt (0,5-25 µM) concentrations. After 48 h incubation in the

presence of 5FU under normoxic or hypoxic conditions (for the last 24 h), cells were

27

harvested and counted using a Beckman Coulter Z series cell counter (Beckman Coulter,

Fullerton, CA, USA).

Clonogenic assay

To compare the sensitivity of monolayers and spheroids to 5FU or OxPt, the clonogenic assay

was used. This in vitro assay is based on the ability of a single cell to grow into a colony. It

essentially tests every cell in the population for its ability to undergo “unlimited” division and

determines whether a cell has undergone reproductive death. Following 48 h treatment with

5FU or OxPt, spheroids or monolayer cultures were disaggregated or detached, respectively,

using a Trypsin-EDTA solution, counted and 200 cells/well were plated onto six well plates

and allowed to grow for 8 d. At the end of this period, cell colonies were fixed with 95% v/v

methanol for 5’ at RT, and stained with a solution of methylene blue 0,05 % for 45’. Only

colonies consisting of more than 50 cells were scored and were expressed as fractions of the

number of colonies in control wells (Fu). IC50 values were calculated by the median effect

equation [73].

Flow cytometric analysis: HIF-1 activity

HIF-1α activity was assessed in HCT116/HRP-EGFP cells grown as monolayer cultures

(under normoxic or hypoxic conditions) or as spheroids, with or without PMX290 (0,5 µM for

monolayers; 2,5 µM for spheroids) for 48 h or EZN-2968 (see above); the effects of EZN-

2968 were compared with those obtained in mock-transfected cells and cells transfected with

the scrambled LNA. At the end of the treatment, cells from monolayers were harvested,

resuspended in PBS and immediately analysed by flow cytometry. Spheroids were

disaggregated as described above, resuspended in PBS and analysed. EGFP fluorescence data

were collected and fluorescence intensity was expressed as mean fluorescence channel

(MFC).

Flow cytometric analysis: apoptosis

Induction of apoptotic cell death under different experimental conditions was also evaluated

by flow cytometry. Monolayer cells were exposed for 48 h to 5FU (10 or 100 µM), or to OxPt

(1 and 10 µM) alone or in combination with PMX290 (0,5 µM, last 48 h), under normoxic or

28

Fig15| Monoparametric

histogram obtained by

cytofluorometric analysis of PI

coloured samples to determine

the percentage of apoptotic cells

(i.e. the sub G1 peak).

hypoxic conditions. The same pattern for HCT116 cells that were transfected wit EZN-2968,

EZN-3088, or with the vehicle (MOCK), and for HCT116 cells that were transduced with the

HIF-1α MUT or WPT vector. Subsequently cells were

harvested by trypsinisation (pooling adherent and

detached cells), washed in PBS, centrifugated, and fixed in

70 % v/v ethanol ( -20°C). Spheroids were incubated for

48 h in the presence of 5FU (0,5 - 1mM), with or without

PMX290 (2,5 µM), disaggregated by trypsinisation and

processed as described for monolayers. After a further

wash with PBS, DNA was stained with 50 µg/ml

propidium iodide (PI) in PBS in the presence of RNAse A

(30 U/ml) at R.T. for at least 30’. All the samples were

analysed with a FACScan flow cytometer (Becton

Dickinson Mountain View, CA, USA), equipped with a 15

mW, 488 nm and an air-cooled argon ion laser. At least 10.000 events were analysed for each

sample and all data were processed using CellQuest software (Becton Dickinson). Fluorescent

emissions of PI were collected through a 575 nm band-pass filter and acquired in log mode

and the percentage of apoptotic cells in each sample was determined based on the sub-G1

peaks detected in monoparametric histograms (Figure 15).

Statistical analysis

Dose-response curves were analysed by non-linear regression analysis using Calcusyn

(Biosoft, Cambridge, MA), which allowed extrapolating drug IC50 values in parental and

transfected cell lines under different experimental conditions. the IC50 is defined as the drug

concentration inducing a 50% decrease in Fu. Student’s t test was used to evaluate the

difference between 5FU/OxPt IC50 values under normoxic versus hypoxic conditions in

monolayer cultures and in monolayers versus 3D cultures. All the other data were analysed by

ANOVA and Bonferroni’s test for multiple comparisons using Prism 4.03 (GraphPad

Software Inc., San Diego, CA, USA).

29

Results

1. Effect of hypoxia on HIF-1α expression

HC T116 HT29

N H N H

HC T116 HT29

N H N H

Figure 16 shows HIF-1α protein levels in HCT116 and HT29, as assessed by western blot

following 24 h incubation under normoxic or hypoxic conditions. An immunoreactive band at

120 kDa (corresponding to the molecular weight of HIF-1α) is present in both cell lines

maintained under hypoxic conditions, whereas under normoxic conditions the protein is

undetectable, due to its rapid degradation in the presence of oxygen.

2. Effect of hypoxia on HIF-1α activity

Figure 17 shows the results of the flow cytometric analysis performed on HCT116 HRP-

EGFP cells, in which EGFP expression is regulated by a promoter responsive to HIF-1. In

this cells, the intensity of the fluorescence emitted by EGFP is directly related to the

transcriptional activity of HIF-1α and increases significantly following 24h incubation under

hypoxic conditions.

Fig. 16| Protein levels of HIF-1α in cell lines

HCT116 and HT29 in normoxic conditions (N)

or hypoxic conditions (H).

Fig. 17| FACS graph representing the

HIF-1α activity in HCT116 HRP-EGFP

cells in normoxia (N) and hypoxia (H). An

increase in HIF-1α activity is clearly

visible in hypoxic circumstances

(i.e. a right shift of the peak).

C

ell-

nu

mb

er

EGFP-fluorescence

30

3. Effect of hypoxia on cellular response to 5FU and OxPt

3.1. Determination of IC50 values for colon cancer cells grown as

monolayers

HCT116 and HT29 cells were treated for 48 h with increasing concentrations of 5FU (1-250

µM) or OxPt (0,5-25 µM) under either normoxic or hypoxic conditions (during the last 24h)

and vital cells were counted to obtain IC50 values (Table 1).

HCT116 HT29

normoxia hypoxia normoxia hypoxia

5FU 7,15 ± 2,69 µM 13,17 ± 2,57 µM 37,4 ± 3,29 µM 95,91 ± 24,66 µM

OxPt 2,20 ± 0,10 µM 3,07 ± 0,83 µM 0,91 ± 0,07 µM 2,20 ± 0,80 µM

HCT116 cells are less responsive to the cytotoxic effects of 5FU under hypoxic conditions

versus normoxic conditions, as indicated by the nearly twofold value of IC50 whereas no

significant differences can be observed in the response to OxPt. In contrast, hypoxic HT29

cells are significantly less sensitive to both drugs as compared with the results obtained under

normoxic conditions. In addition, the cell lines themselves displayed significant differences in

every case, with the exception of OxPt-treated hypoxic cells.

3.2. Determination of HIF-1 activity for HCT116 cells grown as

spheroids versus monolayers

Three-dimensional cultures were obtained from HCT116 and HCT116 HRP/EGFP cells.

Following 7-day incubation in non-adherent conditions, spheroids are formed, with diameters

ranging from 500 to 600 µm (Figure 18). These experiments were not performed on HT29

cells, due to the low transfection efficiency obtained in this cell line.

Table 1| Effect of 5FU and OxPt (48h) on the growth of HCT116 and HT29 cells as monolayers

under hypoxic or normoxic conditions. Mean IC50 values (Mean ± S.E., n = 3). Grey boxes: p < 0,05

versus normoxic values. Green marked values: p < 0,05 versus HT29 values (in same oxygen

condition and for the same drug).

31

Flow cytometric analysis of cells from HCT116 HRP/EGFP spheroids shows that

fluorescence intensity is significantly higher in these cells than in monolayer cells, indicating

higher HIF1- activity (Figure 19).

3.3. Determination of IC50 values for HCT116 cells grown as spheroids

In agreement with the results obtained in 3.1, 5FU is significantly less potent in inhibiting the

clonogenic potential of cells derived from HCT116 spheroids than these derived from

HCT116 monolayers. Table 2 shows the IC50 values obtained with the clonogenic assay.

Fig. 19| HIF-1 transcriptional activity in HCT116 HRP/EGFP monolayers (grey outline) or 3D-

spheroids (black peak). There is significantly higher fluorescence intensity in spheroids, indicating

a higher HIF-1 activity.

Fig. 18| Seven-day spheroids obtained from HCT116 HRP/EGFP cells (Right; visualised by confocal

microscopy, merged fluorescence and clear field images, with visible HIF-1 activity; more obvious in

the centre of the spheroid) and HCT116 cells (Right; visualised by phase contrast microscopy).

32

HCT116

monolayer spheroid

5FU 12,26 ± 4.76 132.76 ± 41.04

3.4. Determination of apoptosis

Figure 20 present histograms of the percentage of apoptotic cells, normalized for the value

obtained in control samples (T/C: drug Treatment/Control) for HCT116 and HT29 cells in

either hypoxic or normoxic conditions. While OxPt induces apoptosis in HCT116 cells in a

concentration-dependent fashion, no significant differences are detectable between normoxic

and hypoxic conditions (Figure 20a), in agreement with the results presented in Table 1.

In contrast, when HCT116 were treated with 5FU, an increase in apoptotic cells was only

observed following exposure to a 100 µM concentration and this was significantly lower

under hypoxic versus normoxic conditions (Figure 20b).

In HT29 cells, OxPt induces a concentration-dependent increase in apoptotic cells under

normoxic conditions, whereas no effect is observed in hypoxic cells (Figure 20c).

In contrast, no effect is observed, in terms of induction of apoptosis, when HT29 cells are

exposed to 5FU, irrespective of oxygen levels (Figure 20d).

Fig. 20a| Percentages of apoptotic cells (Treatment/Control) in HCT116 cells treated with Oxaliplatin

(1 and 10 µM) for 48 h in either hypoxic or normoxic conditions.

Table 2| Mean IC50 values (Mean ± S.E., n = 3). The HCT116 cells are treated for 48h with 5FU in

either the context of spheroids or monolayers. Grey box: p < 0,001 versus monolayer values.

33

Fig. 20c| Percentages of apoptotic cells (Treatment/Control) in HT29 cells treated with OxPt (1 and

10 µM) for 48 h in either hypoxic or normoxic conditions. Mean ± S.E., n ≥ 3. *** p < 0,001

versus normoxic value.

***

Fig. 20b| Percentages of apoptotic cells (Treatment/Control) in HCT116 cells treated with 5FU (10

and 100 µM) for 48 h under normoxic or hypoxic conditions. Mean ± SE, n ≥ 3; *** p < 0,001.

Fig. 20d| Percentages of apoptotic cells (Treatment/Control) in HT29 cells treated with 5FU (10

and 100 µM) for 48 h in either hypoxic or normoxic conditions. Mean ± S.E., n ≥ 3.

µM

µM

34

4. Effect of modulation of HIF-1α on the response of colon cancer

cells to 5FU and OxPt

To demonstrate the crucial role of HIF-1 in the decreased responsiveness of colon cancer cells

to chemotherapy under hypoxic conditions, different agents were used to downregulate HIF-

1α expression and/or HIF-1 transcriptional activity:

1. PMX290, an inhibitor of the thioredoxin system (TRX), that has been shown to

inhibit HIF-1transcriptional activity [66].

2. EZN2968, a LNA antisense oligonucleotide directly targeting the HIF-1α mRNA [68].

In addition, to confirm the ability of HIF to modulate the cellular response to chemotherapy,

HIF-1α was also upregulated, by infecting the cells with a lentiviral vector encoding a

degradation-resistant HIF-1α mutant form.

4.1. Effect of PMX290 treatment on HIF-1 activity and response to 5FU in

HCT116 cells

Figure 21a shows the effect of 48 h exposure to PMX 0,5 µM under hypoxic conditions on the

fluorescence intensity emitted by HCT116/HRP-EGFP cells; the results obtained for these

cells (C) are compared with those obtained for untreated cells under either normoxic (A) or

hypoxic (B) conditions. Whereas the peak of fluorescence of the hypoxic control (B) is

shifted towards right in comparison with the normoxic conditions (A), treatment with PMX

(C) indeed reduces this shift, i.e. HIF-1 transcriptional activity.

A B

C Fig 21a| Effect of PMX290 on HIF-1

transcriptional activity in HCT116/HRP-EGFP

cells. Peak A: normoxic untreated control. Peak

B: hypoxic untreated control. Peak C: PMX290

(0.5 µM for 48 h) under hypoxic conditions.

35

Figure 21b shows the percentage of apoptotic cells, obtained by flow cytometric analysis of

HCT116 cells exposed to 5FU and PMX290 under normoxic (Left) and hypoxic (Right)

conditions. The results of these experiments confirm that the cells are more resistant to 5FU

under hypoxic versus normoxic conditions and indicate that treatment with PMX290 alone

increases the percentage of apoptotic cells versus untreated controls, under both normoxia and

hypoxia. When the two agents are used in combination, PMX290 also increases the

percentage of apoptotic cells over the values observed for 5FU alone, and, most importantly,

it significantly restores the ability of 5FU to induce apoptosis in hypoxic cells, almost to the

extent observed in normoxia.

Fig. 21b| Percentages of apoptotic HCT116 cells (Treatment/Control) treated with 5FU 100 µM for 48

h with or without PMX290 (0.5 µM) and incubated in normoxic (Left) or hypoxic conditions (Right).

Mean ± S.E., n ≥ 3; ** p < 0,01 versus no PMX; * p < 0,05 versus no PMX; *** p < 0,001 versus no

PMX.

4.2. Effect of PMX290 treatment on the response of HT29 cells to OxPt and

5FU

The histograms in figure 22 represent the percentage of apoptotic HT29 cells following

treatment with OxPt (1 or 10 µM) for 48 h, with or without PMX290 (0,5 µM), under either

hypoxia or normoxia, in comparison with control (untreated) cells. In contrast to HCT116

cells, HT29 cells do not significantly respond to PMX290 alone, and PMX is unable to

modify the response of these cells to OxPt under normoxic conditions; however, treatment of

hypoxic cells with PMX290 restores the response as achieved in normoxic levels concerning

the 10 µM OxPt treatment.

36

Fig 22| Percentages of apoptotic HT29 cells (Treatment/Control) treated with OxPt 1 or 10 µM for 48

h with or without PMX290 0.5 µM and incubated in normoxia (Left) or in hypoxia (Right). Mean ±

S.E. ≥ 2; *** p < 0,001 versus no PMX.

Similar results are also observed when PMX290 is combined with 5FU (Figure 23).

Fig 23| Percentages of apoptotic HT29 cells (Treatment/Control) following treatment with 5FU 10 or

100 µM for 48 h with or without PMX290 0.5 µM and incubated in normoxia (Left) or in hypoxia

(Right). Mean ± S.E., n ≥ 2; * p < 0,05 versus no PMX.

4.3. Effect of PMX290 treatment on HIF-1 activity and apoptotic response to

5FU in HCT116 cells grown as spheroids

Treatment of HCT116-HRP cells grown as spheroids with PMX290 (2,5 µM for 48 h)

induces a significant reduction in HIF-1 activity. In the histogram (Figure 24), where two cell

37

subpopulations are visible, indicating that a significant fraction of cells has shifted towards

lower fluorescence intensities thus has a significant decrease in HIF-1 activity.

Flow cytometric analysis of apoptotic cells from spheroids exposed to 5FU with or without

PMX290 (Figure 25), shows that combined exposure to the quinol (2,5 µM) causes a

significant increase in apoptosis induction by 5FU (0,5 - 1 µM for 48 h) versus exposure to

5FU alone.

0 0.5 1.0 0

5

10

15

* *

no PMX290

PMX290 2.5 µM

µM 5FU

% A

po

pto

tic c

ell

s

Fig. 24| Flow cytometric analysis of HCT116/HRP-EGFP spheroids without PMX290 treatment

(grey outline) and HCT116/HRP-EGFP spheroids treated with PMX290 (black outline).

EGFP-FLUORESCENCE

Fig. 25| 5FU-induced apoptosis in HCT116/HRP-EGFP spheroid-derived cells. Mean ± S.E.,

n = 3; * p < 0.05 versus cells from spheroids treated with 5FU alone. Empty bars: no PMX290;

filled bars: 2.5 mM PMX290 for 48h.

38

5. Effect of EZN-2968 treatment on HIF-1α expression, HIF-1

activity and apoptotic response to 5FU in HCT116 cells

Figure 26 shows that EZN2968 inhibits HIF-1α expression in HCT116 cells following 24h

incubation under hypoxic conditions. A scrambled (SCR) LNA oligonucleotide, containing

the same nucleotides in random sequence, was used as a negative control.

Figure 27 shows the results of the flow cytometric analysis of HIF-1α activity in HCT116-

HRP cells transfected with EZN-2969 or with the scrambled oligonucleotide: under normoxic

conditions no significant difference can be seen between the fluorescent signals emitted by

cells transfected with the two oligonucleotides. However, under hypoxic conditions the

fluorescence detected in the SCR-transfected cells is significantly higher as compared with

cells transfected with EZN-2968.

Figure 28 shows the apoptotic effect of 5FU (10 and 100 µM for 48 h) on HCT116 cells

(Treatment/Control) transfected with EZN-2968 or with the scrambled control

SCR SCR

NORM HYPO

2968 2968

Fig. 26| Western blot of HCT116 cell lysates that

were transfected with the A.S.O. EZN2968 10 nM

in normoxic (Left) and hypoxic (Right) conditions.

SCR = scrambled sequence that functions as a

negative control.

Fig. 27| Effect of EZN-2968 (HIF -) on HIF-1 activity in HCT116/HRP-EGFP cells under either

normoxic or hypoxic conditions. Mean ± S.E., n ≥ 2; * p < 0,05 versus normoxia and SCR;

° p < 0,05 versus normoxia.

FL

UO

RE

SC

EN

CE

39

oligonucleotide under normoxic or hypoxic conditions (for the last 24h). Mock-transfected

cells, i.e. HCT116 cells treated only with the transfection reagent without oligonucleotides,

were also used as additional controls (not shown in the figure). The results indicate that both

mock-transfected cells and cells transfected with the scrambled oligonucleotide are less

responsive to 5FU treatment under hypoxic conditions, whereas cells transfected with

EZN2968 exhibit enhanced sensitivity in response to 5FU, as indicated by a significant

increase in the percentage of apoptotic cells as compared to cells transfected with the control

oligonucleotide under the same conditions.

6. Effects of the expression of a degradation-resistant variant of

HIF-1α on the response of HCT116 cells to 5FU

To confirm that HIF-1 is indeed responsible for the observed diminished response of HCT116

cells to 5FU under hypoxic conditions, HCT116 cells were infected with lentiviral vectors

encoding a mutant, degradation-resistant variant of HIF-1α. The cell line obtained, called

HCT116/HIF-1α MUT, was compared with HCT116 cells transduced with the control vector

WPT and showed a marked increase in HIF-1α protein levels under normoxic conditions

(Figure 29a).

Fig. 28| Induction of apoptosis (Treatment/Control) by 5FU (10 or 100 µM for 48 h) in HCT116

cells transfected with either EZN2968 (hif-) or with the scrambled oligonucleotide (scrambled)

and incubated in either hypoxia or normoxia. Mean ± S.E., n ≥ 3; ** p < 0,01 versus normoxia.

**

40

vector

HIF1αMUT

HIF-1α

vector

HIF1αMUT

HIF-1α

In Table 3 the IC50 values obtained for the two cell lines following treatment with 5FU are

reported, indicating that increased expression of mutant HIF-1α inhibits the cytotoxic activity

of 5FU. HCT116/HIF-1α MUT cells are also refractory to the proapoptotic effect of 5FU, as

indicated by the observation that even relatively high drug concentrations (i.e. 250 – 500 µM)

do not induce a significant increase in apoptotic cells, in contrast with HCT116 WPT cells,

that undergo apoptosis in a concentration-dependent fashion (Figure 29b).

Table 3| IC50 values obtained following 48 h exposure to 5FU under normoxic conditions. Mean ±

S.E., n ≥ 3; p < 0,001.

Normoxia

HCT116/HIF-1α MUT HCT116 WPT

5FU 658,58 ± 62,2 µM 6,55 ± 1,46 µM

Fig. 29a| Effect of HCT 116 cell transduction

with the HIF-1αMUT or WPT (control) lentiviral

vectors under normoxic conditions on HIF-1α protein

levels.

Fig 29b| Effect of overexpression of a degradation-resistant HIF-1α form on 5FU induced

apoptosis. Mean ± S.E., n ≥ 3; * p < 0.05 versus untreated HCT116/WPT cells; ** p < 0.001 versus

untreated HCT116/WPT cells; ° p < 0.01 versus HCT116/WPT cells at the same 5FU concentration.

41

Discussion

We utilised two different cell lines each with specific characteristics and two

chemotherapeutic drugs, OxPt and 5FU, both used in the first-line treatment of advanced

colorectal cancer. Furthermore HIF-1 modulating agents (potential future drugs) were added

to further clarify the importance of HIF-1 in the obtained results.

5-Fluorouracil

Our data shows that HCT116 cells are more sensitive to 5FU than HT29 cells (for both

oxygen conditions significantly, regarding Table 1), and both become more resistant when

they are incubated under hypoxic conditions.

Why are HT29 cells more resistant to 5FU than HCT116 cells? We know that these

cell lines differ in some important aspects potentially involved in cell response to 5FU. i)

HCT116 cells express a functional p53 protein, whereas HT29 cells carry an inactivating

R237H mutation [69]. Lack of functional p53 protein in HT29 cells may negatively affect

their ability to undergo apoptosis following 5FU treatment, due to reduced expression of pro-

apoptotic proteins (e.g. Bax). This hypothesis is supported by the findings of Boyer et al.,

who demonstrated a five-fold lower IC50 for 5FU in HCT116 p53 wild-type (p53+/+

) cells as

compared to an isogenic p53-null (p53-/-

) cell line [74]. ii) Peters GJ et al. have observed that

p53, or its lack, can affect the feedback mechanism regulating TS protein synthesis following

5FU treatment, whereby free TS can repress the translation of its own mRNA, whereas

binding to 5FU causes de-repression of translation. The finding that colon carcinoma cells

expressing a mutant (mt) p53 exhibit a higher induction of TS than p53-proficient suggests

that p53 might participate to the downregulation of TS at this level; as TS is the major target

for 5FU activity, this effect might thus contribute to the observed differences in response to

5FU between p53+/+

and p53-/-

cells cells [75]. iii) Concerning the number of tandem repeats

of a 28 bp sequence in the enhancer region at the 5’UTR of the TS gene, a well known

polymorphism leading to different levels of TS, both HCT116 and HT29 cells have a

homozygous wildtype genotype (2R/2R) [76]. However, in spite of this common genotypic

feature, Nief et al. have demonstrated that TS expression is higher in HCT116 than in HT29

cells (in a 23,4 : 15,1 ratio), and the ratio between TS activity in the two cell lines is even

higher (39,6 : 2,3 respectively), indicating that other factors are involved in determining this

divergence [77]. iv) In this regard, HCT116 and HT29 cells have been shown to differ for

42

another common polymorphism, consisting in a 6 bp deletion in the 3’UTR of the TS gene,

which has been associated with reduced stability and translation of the TS mRNA. In contrast

with HT29 cells, HCT116 cells exhibit this deletion in homozygous form, and this might

contribute to the higher 5FU sensitivity exhibited by this cell line [77]. v) In addition,

HCT116 cells are known to express undetectable levels of DPD, the major 5FU-inactivating

enzyme [78], and clearly detectable levels of thymidine phosphorylase (TP), one of the

enzymes concurring to anabolic activation of 5FU; these two facts could contribute to the

higher sensitivity to 5FU exhibited by this cell line, at least in comparison with HT29 cells,

which only express low levels of TP activity [71], while exhibiting measurable levels of DPD

expression and activity [79].

All the 5FU-sensitivity modifying properties above described (i-v) do not seem to

have a significant impact on the effect of hypoxia on drug response. The mechanism

underlying decreased drug response under hypoxia is very likely multifactorial, involving

both HIF-1-dependent and -independent effects. In our experimental models, HIF-1 clearly

makes a major contribution to the resistance of hypoxic HCT116 cells to 5FU as shown by

different orders of evidence. First, incubation under hypoxic conditions leads to accumulation

of HIF-1α protein and to an increase in HIF-1 transcriptional activity, as assessed in HCT116

cells stably transfected with a reporter plasmid expressing EGFP under the control of multiple

copies of the HRE derived from the human VEGF gene promoter. Second, HIF-1α

knockdown by transfection of HCT116 cells with the antisense oligonucleotide EZN-2968,

that specifically targets HIF-α mRNA, partially prevents the development of resistance to

5FU under hypoxic conditions. Finally, infection of HCT116 cells with a lentiviral vector

encoding a degradation-resistant form of HIF-1α, lacking the two proline residues that are

crucial for HIF-1α oxygen-dependent hydroxylation and subsequent degradation, leads to

resistance to 5FU under normoxic conditions. In HT29 cells, hypoxia-induced resistance to

the cytotoxic effect of 5FU is also associated with HIF-1α accumulation; however, due to the

low transfection efficiencies obtained with this cell line for both oligonucleotides and

lentiviral vector, it was impossible to assess HIF-1 activity. The observation that treatment

with PMX290 (which inhibits HIF-1 activity, but not HIF-1α accumulation, see below)

partially restores the sensitivity of HT29 cells to 5FU indirectly supports the hypothesis that

increased HIF-1 activity also contributes to 5FU resistance in this cell line.

Why are hypoxic HCT116 and HT29 cells less responsive than normoxic cells to

5FU? Hypoxic cells have the propensity to acquire genetic abnormalities that confer a ‘more

malignant’ phenotype, such as increased expression of the drug exporting proteins MDR1 or

43

BRCP, or proteins that participate in thiol-mediated drug detoxication [51, 80, 81]. However

5FU is not a known substrate for either transporter, and therefore this mechanism is not likely

to play a major role in 5FU resistance. Impairment of mismatch repair activity could be

involved in hypoxia-induced resistance to a number of agents requiring activation of this

DNA repair system to effectively generate a death signal, including 5FU [82, 83]. We know

that HCT116 cells are intrinsically defective in mismatch repair due to a homozygous

mutation in hMLH1, hence they exhibit MSI; however, this is not the case for HT29 cells. In

addition, Meyers et al. have demonstrated that following 5FU treatment there are no

significant differences in apoptosis between MMR-deficient cells (i.e. HCT116) in

comparison with isogenic MMR-proficient cells (i.e. HCT116 3-6) [29]. Thus, as confirmed

by several studies, we cannot consider the status of MMR genes as a major factor determining

the response to 5FU in our cell lines [84]. The most likely hypothesis to explain the reduced

cytotoxicity of 5FU in hypoxic HCT116 and HT29 cells involves critical alterations in the

levels of pro- and antiapoptotic factors. Increasing evidence indicates that HIF-1 activation

modulates the expression of pro- and antiapoptotic genes; for instance, significantly increased

levels of survivin and Bcl-xL, and decreased levels of Bid and Bax were reported [85, 55, 47].

An imbalance between pro-and antiapoptotic signals favouring cell survival could very well

account for a reduced response to a wide range of anticancer agents and could explain the

observed 5FU resistance under hypoxic conditions.

Having established the role of HIF-1 in 5FU resistance, we assessed the ability of

PMX290 to restore the response of our cell lines to 5FU. PMX290 has been shown to

decrease HIF-1α expression and/or HIF-1 activity due to its ability to inhibit Trx-1, a positive

modulator of HIF-1 [66]. Our results indicate that PMX290 decreases HIF-1 activity in

HCT116HRP cells grown both as monolayers under hypoxic conditions and as three-

dimensional spheroids at normoxic pO2 values. Interestingly, combining PMX290 with 5FU

significantly enhances the antiproliferative effect of 5FU in both cell lines tested (as assessed

by cytotoxicity assays) and significantly increases the percentage of apoptotic cells as

compared with 5FU alone. The results obtained on spheroids confirm the results obtained in

monolayers. Flow cytometric analysis of spheroids obtained from HCT116 HRP/EGFP cells,

indicates that hypoxia develops spontaneously within the cell mass, leading to HIF-1

activation (increased fluorescence is visible at the centre of the spheroid, see Figure 18). The

presence of hypoxic regions causes a significant decrease in the antiproliferative effect of

5FU, as assessed by the clonogenic assays on disaggregated spheroids in comparison with

monolayer cells. However, it should not be neglected that limited diffusion of the drugs into

44

the centre of the spheroid could also contribute to the 10 fold greater IC50 value obtained for

spheroids.

The observation that subtoxic concentrations of PMX290 simultaneously reduce HIF-1

activity and enhance cell response to 5FU indicates that HIF-1 inhibition could be an effective

approach to cell sensitisation in the clinical management of hypoxic tumours.

Oxaliplatin

Significant differences in cell response between HCT116 and HT29 under normoxic

conditions were also observed with OxPt (Table 1); however, in contrast with 5FU, in this

case HT29 cells appear to be more sensitive than HCT116 to the platinum derivative. What

possible mechanism could account for the observed difference?

Reports on the role played by p53 are highly controversial one. Arango et al. found that the

mutational status of the tumour suppressor gene could not predict the apoptotic response to 10

µM OxPt treatment in a panel of 30 different colorectal cancer cell lines, suggesting that

additional factors modulate sensitivity to this agent [86]. The bulk of clinical data regarding

p53 status and sensitivity to platinum compounds has focused on the first generation

compound cisplatin. However, Howels et al. reported that exposure of wild-type p53 HCT116

cells to OxPt results in increased levels of p53, with subsequent upregulation of the p21 gene,

a major target for p53 transcriptional activity [87]. Increased p53 levels can enhance apoptosis

trough different mechanisms, including Bax upregulation, but they also induce cell cycle

arrest, which favors DNA damage repair and could cause failure of OxPt treatment in p53+/+

HCT116 cells, whereas cannot occur in p53 defective HT29 cells. Moreover, Arango et al.

have reported that the concentration of OxPt necessary to cause a 50% growth inhibition after

72 h of exposure was four-fold higher in HCT116 p53-/-

cells compared to parental isogenic

HCT116 cells, which seems to rule out a protective antiapoptotic role of p53 in this cell line.

[86]. However, these findings in HCT116 cells cannot be extended to HT29 cells. In fact,

Howells et al. have pointed out that survival signalling in response to OxPt exhibits cell line

specificity; HCT116 cells require p53 for the OxPt induced apoptotic response, whereas

mutated p53 in HT29 cells does not prevent this chemotherapy-induced apoptosis [87].

Another aspect differentiating HCT116 from HT29 cells is MMR status, as HCT116 cells

are deficient in this regard, whereas HT29 cells are not [29]. MMR status is definitely

considered as a major determinant of cell response to cisplatin and carboplatin, but for OxPt

45

this has been shown not to be the case; loss of MMR enzyme activity has been demonstrated

not to be involved in OxPt resistance [88].

A third possible difference is the tetraploïd character of HT29 cells as this might require

more OxPt to reach the same degree of genotoxic damage in comparison with 2n cells such as

HCT116; however, our results seem to point in the opposite direction, so while this feature

can ultimately affect the outcome of OxPt treatment, it does not help explain the observed

difference between HCT116 and HT29 cells.

As already observed for 5FU, HT29 incubated under hypoxic conditions are more

resistant than their normoxic counterparts to the cytotoxic action of OxPt; however, no

significant differences were observed for HCT116 cells, so that we can conclude that hypoxia

only plays a minor role in HCT116 resistance against OxPt.

As already noted in the case of 5FU, the mechanism underlying decreased OxPt response in

hypoxia HT29 cells is very likely multifactorial, involving both HIF-1-dependent and

-independent effects. Unfortunately, direct measures of HIF-1 activity in HT29 cells were not

possible; however, the hypothesis that HIF-1 plays a role in the reduced response to OxPt is

supported by the results following combined treatment with PMX290, as this HIF-1-inhibiting

agent significantly reduces the resistant state observed in hypoxic HT29 cells.

Further investigations will be necessary to identify genes that are predictive of cell

response to OxPt and that may be affected by the HIF-1 transcription factor. At this moment

we can note for example that high expression levels of the nucleotide excision repair gene

ERCC1 predict poor response to OxPt [74]. Another example is the ATP-binding cassette

half-transporter BCRP/ATP-binding cassette G2; Boyer et al. demonstrated overexpression of

this efflux pump in both p53+/+

and p53-/-

OxPt resistant cell lines [74]. Cisplatin is not

known to act as a substrate for BCRP, and no clear evidence has been reported that OxPt may

act as such, but given the structural differences, it might be. Interestingly, though, BCRP

expression has been shown to be upregulated under hypoxic conditions through a HIF-1-

dependent mechanism, so the role of this transporter in hypoxia-induced resistance is

certainly worth investigating [80]. Expression levels of other transport proteins, such as the

copper transporter hCtr1 and the organic cation transporter hOCT1 (these are certainly OxPt

transporters), have been correlated to cytotoxicity of OxPt in HCT116 cells and can also

represent additional factors determining increased OxPt resistance in this cell line in

comparison with HT29 cells [89].

46

In summary, while OxPt resistance is generally attributed to decreased cellular uptake

and/or increased efflux (i.e. intracellular accumulation is detrimental) and to efficient repair of

DNA-Pt adducts, it is not clear how these factors might contribute to hypoxia in OxPt

resistance [89, 90]. It may be important to emphasise that an highly selective hypoxic

environment can facilitate mutational events (such as p53 loss of function) and adaptive

responses (increased resistance) that can decrease susceptibility to both hypoxia and OxPt

induced cell death [20, 91]

General conclusion

To conclude, our data indicate that 1) HIF-1 activation may play an important role in the

response of colorectal cancer cell to 5FU and OxPt, and is in fact a major determinant of

hypoxia-induced drug resistance; 2) agents inhibiting HIF-1 could help improve the

therapeutic index of cytotoxic agents currently used in the clinical management of colorectal

cancer; and 3) the genetic makeup of the individual tumour may affect its response to hypoxia

and its susceptibility to hypoxia-induced resistance, as demonstrated by the different response

observed in HCT116 and HT29 cells, once more underscoring the importance of tumor

genetic profiling in directing the choice of therapeutic options.

47

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